Isolated human transporter proteins, nucleic acid molecules encoding human transporter proteins,and uses thereof

ABSTRACT

The present invention provides amino acid sequences of peptides that are encoded by genes within the human genome, the transporter peptides of the present invention. The present invention specifically provides isolated peptide and nucleic acid molecules, methods of identifying orthologs and paralogs of the transporter peptides, and methods of identifying modulators of the transporter peptides.

RELATED APPLICATIONS

[0001] The present application claims priority to provisionalapplications U.S. Ser. No. 60/256,339 filed Dec. 19, 2000 (Atty. DocketCL001051-PROV).

FIELD OF THE INVENTION

[0002] The present invention is in the field of transporter proteinsthat are related to the vacuolar ATPase transporter subfamily,recombinant DNA molecules, and protein production. The present inventionspecifically provides novel peptides and proteins that effect ligandtransport and nucleic acid molecules encoding such peptide and proteinmolecules, all of which are useful in the development of humantherapeutics and diagnostic compositions and methods.

BACKGROUND OF THE INVENTION Transporters

[0003] Transporter proteins regulate many different functions of a cell,including cell proliferation, differentiation, and signaling processes,by regulating the flow of molecules such as ions and macromolecules,into and out of cells. Transporters are found in the plasma membranes ofvirtually every cell in eukaryotic organisms. Transporters mediate avariety of cellular functions including regulation of membranepotentials and absorption and secretion of molecules and ion across cellmembranes. When present in intracellular membranes of the Golgiapparatus and endocytic vesicles, transporters, such as chloridechannels, also regulate organelle pH. For a review, see Greger, R.(1988) Annu. Rev. Physiol. 50:111-122.

[0004] Transporters are generally classified by structure and the typeof mode of action. In addition, transporters are sometimes classified bythe molecule type that is transported, for example, sugar transporters,chlorine channels, potassium channels, etc. There may be many classes ofchannels for transporting a single type of molecule (a detailed reviewof channel types can be found at Alexander, S. P. H. and J. A. Peters:Receptor and transporter nomenclature supplement. Trends Pharmacol.Sci., Elsevier, pp. 65-68 (1997) andhttp://www-biology.ucsd.edu/˜msaier/transport/titlepage2.html.

[0005] The following general classification scheme is known in the artand is followed in the present discoveries.

[0006] Channel-type transporters. Transmembrane channel proteins of thisclass are ubiquitously found in the membranes of all types of organismsfrom bacteria to higher eukaryotes. Transport systems of this typecatalyze facilitated diffusion (by an energy-independent process) bypassage through a transmembrane aqueous pore or channel without evidencefor a carrier-mediated mechanism. These channel proteins usually consistlargely of a-helical spanners, although b-strands may also be presentand may even comprise the channel. However, outer membrane porin-typechannel proteins are excluded from this class and are instead includedin class 9.

[0007] Carrier-type transporters. Transport systems are included in thisclass if they utilize a carrier-mediated process to catalyze uniport (asingle species is transported by facilitated diffusion), antiport (twoor more species are transported in opposite directions in a tightlycoupled process, not coupled to a direct form of energy other thanchemiosmotic energy) and/or symport (two or more species are transportedtogether in the same direction in a tightly coupled process, not coupledto a direct form of energy other than chemiosmotic energy).

[0008] Pyrophosphate bond hydrolysis-driven active transporters.Transport systems are included in this class if they hydrolyzepyrophosphate or the terminal pyrophosphate bond in ATP or anothernucleoside triphosphate to drive the active uptake and/or extrusion of asolute or solutes. The transport protein may or may not be transientlyphosphorylated, but the substrate is not phosphorylated.

[0009] PEP-dependent, phosphoryl transfer-driven group translocators.Transport systems of the bacterial phosphoenolpyruvatc:sugarphosphotransferase system are included in this class. The product of thereaction, derived from extracellular sugar, is a cytoplasmicsugar-phosphate.

[0010] Decarboxylation-driven active transporters. Transport systemsthat drive solute (e.g., ion) uptake or extrusion by decarboxylation ofa cytoplasmic substrate are included in this class.

[0011] Oxidoreduction-driven active transporters. Transport systems thatdrive transport of a solute (e.g., an ion) energized by the flow ofelectrons from a reduced substrate to an oxidized substrate are includedin this class.

[0012] Light-driven active transporters. Transport systems that utilizelight energy to drive transport of a solute (e.g., an ion) are includedin this class.

[0013] Mechanically-driven active transporters. Transport systems areincluded in this class if they drive movement of a cell or organelle byallowing the flow of ions (or other solutes) through the membrane downtheir electrochemical gradients.

[0014] Outer-membrane porins (of b-structure). These proteins formtransmembrane pores or channels that usually allow the energyindependent passage of solutes across a membrane. The transmembraneportions of these proteins consist exclusively of b-strands that form ab-barrel. These porin-type proteins are found in the outer membranes ofGram-negative bacteria, mitochondria and eukaryotic plastids.

[0015] Methyltransferase-driven active transporters. A singlecharacterized protein currently falls into this category, theNa+-transporting methyltetrahydromethanopterin:coenzyme Mmethyltransferase.

[0016] Non-ribosome-synthesized channel-forming peptides or peptide-likemolecules. These molecules, usually chains of L- and D-amino acids aswell as other small molecular building blocks such as lactate, formoligomeric transmembrane ion channels. Voltage may induce channelformation by promoting assembly of the transmembrane channel. Thesepeptides are often made by bacteria and fungi as agents of biologicalwarfare.

[0017] Non-Proteinaceous Transport Complexes. Ion conducting substancesin biological membranes that do not consist of or are not derived fromproteins or peptides fall into this category.

[0018] Functionally characterized transporters for which sequence dataare lacking. Transporters of particular physiological significance willbe included in this category even,though a family assignment cannot bemade.

[0019] Putative transporters in which no family member is an establishedtransporter. Putative transport protein families are grouped under thisnumber and will either be classified elsewhere when the transportfunction of a member becomes established, or will be eliminated from theTC classification system if the proposed transport function isdisproven. These families include a member or members for which atransport function has been suggested, but evidence for such a functionis not yet compelling.

[0020] Auxiliary transport proteins. Proteins that in some wayfacilitate transport across one or more biological membranes but do notthemselves participate directly in transport are included in this class.These proteins always function in conjunction with one or more transportproteins. They may provide a function connected with energy coupling totransport, play a structural role in complex formation or serve aregulatory function.

[0021] Transporters of unknown classification. Transport proteinfamilies of unknown classification are grouped under this number andwill be classified elsewhere when the transport process and energycoupling mechanism are characterized. These families include at leastone member for which a transport function has been established, buteither the mode of transport or the energy coupling mechanism is notknown.

Ion Channels

[0022] An important type of transporter is the ion channel. Ion channelsregulate many different cell proliferation, differentiation, andsignaling processes by regulating the flow of ions into and out ofcells. Ion channels are found in the plasma membranes of virtually everycell in eukaryotic organisms. Ion channels mediate a variety of cellularfunctions including regulation of membrane potentials and absorption andsecretion of ion across epithelial membranes. When present inintracellular membranes of the Golgi apparatus and endocytic vesicles,ion channels, such as chloride channels, also regulate organelle pH. Fora review, see Greger, R. (1988) Annu. Rev. Physiol. 50:111-122.

[0023] Ion channels are generally classified by structure and the typeof mode of action. For example, extracellular ligand gated channels(ELGs) are comprised of five polypeptide subunits, with each subunithaving 4 membrane spanning domains, and are activated by the binding ofan extracellular ligand to the channel. In addition, channels aresometimes classified by the ion type that is transported, for example,chlorine channels, potassium channels, etc. There may be many classes ofchannels for transporting a single type of ion (a detailed review ofchannel types can be found at Alexander, S. P. H. and J. A. Peters(1997). Receptor and ion channel nomenclature supplement. TrendsPharmacol. Sci., Elsevier, pp. 65-68 andhttp://www-biology.ucsd.edu/˜msaier/transport/toc.html.

[0024] There are many types of ion channels based on structure. Forexample, many ion channels fall within one of the following groups:extracellular ligand-gated channels (ELG), intracellular ligand-gatedchannels (ILG), inward rectifying channels (INR), intercellular (gapjunction) channels, and voltage gated channels (VIC). There areadditionally recognized other channel families based on ion-typetransported, cellular location and drug sensitivity. Detailedinformation on each of these, their activity, ligand type, ion type,disease association, drugability, and other information pertinent to thepresent invention, is well known in the art.

[0025] Extracellular ligand-gated channels, ELGs, are generallycomprised of five polypeptide subunits, Unwin, N. (1993), Cell 72:31-41; Unwin, N. (1995), Nature 373: 37-43; Hucho, F., et al., (1996) J.Neurochem. 66: 1781-1792; Hucho, F., et al., (1996) Eur. J. Biochem.239: 539-557; Alexander, S. P. H. and J. A. Peters (1997), TrendsPharmacol. Sci., Elsevier, pp. 4-6; 36-40; 42-44; and Xue, H. (1998) J.Mol. Evol. 47: 323-333. Each subunit has 4 membrane spanning regions:this serves as a means of identifying other members of the ELG family ofproteins. ELG bind a ligand and in response modulate the flow of ions.Examples of ELG include most members of the neurotransmitter-receptorfamily of proteins, e.g., GABAI receptors. Other members of this familyof ion channels include glycine receptors, ryandyne receptors, andligand gated calcium channels.

The Voltage-Gated Ion Channel (VIC) Superfamily

[0026] Proteins of the VIC family are ion-selective channel proteinsfound in a wide range of bacteria, archaea and eukaryotes Hille, B.(1992), Chapter 9: Structure of channel proteins; Chapter 20: Evolutionand diversity. In: Ionic Channels of Excitable Membranes, 2nd Ed.,Sinaur Assoc. Inc., Pubs., Sunderland, Mass.; Sigworth, F. J. (1993),Quart. Rev. Biophys. 27: 1-40; Salkoff, L. and T. Jegla (1995), Neuron15: 489-492; Alexander, S. P. H. et al., (1997), Trends Pharmacol. Sci.,Elsevier, pp. 76-84; Jan, L. Y. et al., (1997), Annu. Rev. Neurosci. 20:91-123; Doyle, D. A, et al., (1998) Science 280: 69-77; Terlau, H. andW. Stuthmer (1998), Naturwissenschaften 85: 437-444. They are oftenhomo- or heterooligomeric structures with several dissimilar subunits(e.g., a1-a2-d-b Ca²⁺ channels, ab₁b₂ Na⁺ channels or (a)₄-b K⁺channels), but the channel and the primary receptor is usuallyassociated with the a (or a1) subunit. Functionally characterizedmembers are specific for K⁺, Na⁺ or Ca²⁺. The K⁺ channels usuallyconsist of homotetrameric structures with each a-subunit possessing sixtransmembrane spanners (TMSs). The al and a subunits of the Ca²⁺ and Na⁺channels, respectively, are about four times as large and possess 4units, each with 6 TMSs separated by a hydrophilic loop, for a total of24 TMSs. These large channel proteins form heterotetra-unit structuresequivalent to the homotetrameric structures of most K⁺ channels. Allfour units of the Ca²⁺ and Na⁺ channels are homologous to the singleunit in the homotetrameric K⁺ channels. Ion flux via the eukaryoticchannels is generally controlled by the transmembrane electricalpotential (hence the designation, voltage-sensitive) although some arecontrolled by ligand or receptor binding.

[0027] Several putative K⁺-selective channel proteins of the VIC familyhave been identified in prokaryotes. The structure of one of them, theKcsA K⁺ channel of Streptomyces lividans, has been solved to 3.2 Åresolution. The protein possesses four identical subunits, each with twotransmembrane helices, arranged in the shape of an inverted teepee orcone. The cone cradles the “selectivity filter” P domain in its outerend. The narrow selectivity filter is only 12 Å long, whereas theremainder of the channel is wider and lined with hydrophobic residues. Alarge water-filled cavity and helix dipoles stabilize K⁺ in the pore.The selectivity filter has two bound K⁺ ions about 7.5 Å apart from eachother. Ion conduction is proposed to result from a balance ofelectrostatic attractive and repulsive forces.

[0028] In eukaryotes, each VIC family channel type has several subtypesbased on pharmacological and electrophysiological data. Thus, there arefive types of Ca²⁺ channels (L, N, P, Q and T). There are at least tentypes of K⁺ channels, each responding in different ways to differentstimuli: voltage-sensitive [Ka, Kv, Kvr, Kvs and Ksr], Ca²⁺-sensitive[BK_(Ca), IK_(Ca) and SK_(Ca)] and receptor-coupled [K_(M) and K_(ACh)].There are at least six types of Na⁺ channels (I, II, III, μ1, H1 andPN3). Tetrameric channels from both prokaryotic and eukaryotic organismsare known in which each a-subunit possesses 2 TMSs rather than 6, andthese two TMSs are homologous to TMSs 5 and 6 of the six TMS unit foundin the voltage-sensitive channel proteins. KcsA of S. lividans is anexample of such a 2 TMS channel protein. These channels may include theK_(Na) (Na⁺-activated) and K_(vol) (cell volume-sensitive) K⁺ channels,as well as distantly related channels such as the Tok1 K⁺ channel ofyeast, the TWIK-1 inward rectifier K⁺ channel of the mouse and theTREK-1 K⁺ channel of the mouse. Because of insufficient sequencesimilarity with proteins of the VIC family, inward rectifier K⁺ IRKchannels (ATP-regulated; G-protein-activated) which possess a P domainand two flanking TMSs are placed in a distinct family. However,substantial sequence similarity in the P region suggests that they arehomologous. The b, g and d subunits of VIC family members, when present,frequently play regulatory roles in channel activation/deactivation.

The Epithelial Na⁺ Channel (ENaC) Family

[0029] The ENaC family consists of over twenty-four sequenced proteins(Canessa, C. M., et al., (1994), Nature 367: 463-467, Le, T. and M. H.Saier, Jr. (1996), Mol. Membr. Biol. 13: 149-157; Garty, H. and L. G.Palmer (1997), Physiol. Rev. 77: 359-396; Waldmann, R., et al., (1997),Nature 386: 173-177; Darboux, I., et al., (1998), J. Biol. Chem. 273:9424-9429; Firsov, D., et al., (1998), EMBO J. 17: 344-352; Horisberger,J.-D. (1998). Curr. Opin. Struc. Biol. 10: 443-449). All are fromanimals with no recognizable homologues in other eukaryotes or bacteria.The vertebrate ENaC proteins from epithelial cells cluster tightlytogether on the phylogenetic tree: voltage-insensitive ENaC homologuesare also found in the brain. Eleven sequenced C. elegans proteins,including the degenerins, are distantly related to the vertebrateproteins as well as to each other. At least some of these proteins formpart of a mechano-transducing complex for touch sensitivity. Thehomologous Helix aspersa (FMRF-amide)-activated Na⁺ channel is the firstpeptide neurotransmitter-gated ionotropic receptor to be sequenced.

[0030] Protein members of this family all exhibit the same apparenttopology, each with N- and C-termini on the inside of the cell, twoamphipathic transmembrane spanning segments, and a large extracellularloop. The extracellular domains contain numerous highly conservedcysteine residues. They are proposed to serve a receptor function.

[0031] Mammalian ENaC is important for the maintenance of Na⁺ balanceand the regulation of blood pressure. Three homologous ENaC subunits,alpha, beta, and gamma, have been shown to assemble to form the highlyNa +-selective channel. The stoichiometry of the three subunits isalpha₂, betal, gammal in a heterotetrameric architecture.

The Glutamate-Gated Ion Channel (GIC) Family of NeurotransmitterReceptors

[0032] Members of the GIC family are heteropentameric complexes in whicheach of the 5 subunits is of 800-1000 amino acyl residues in length(Nakanishi, N., et al, (1990), Neuron 5: 569-581; Unwin, N. (1993), Cell72: 31-41; Alexander, S. P. H. and J. A. Peters (1997) Trends Pharmacol.Sci., Elsevier, pp. 36-40). These subunits may span the membrane threeor five times as putative a-helices with the N-termini (theglutamate-binding domains) localized extracellularly and the C-terminilocalized cytoplasmically. They may be distantly related to theligand-gated ion channels, and if so, they may possess substantialb-structure in their transmembrane regions. However, homology betweenthese two families cannot be established on the basis of sequencecomparisons alone. The subunits fall into six subfamilies: a, b, g, d, eand z.

[0033] The GIC channels are divided into three types: (1)a-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-, (2) kainate-and (3) N-methyl-D-aspartate (NMDA)-selective glutamate receptors.Subunits of the AMPA and kainate classes exhibit 35-40% identity witheach other while subunits of the NMDA receptors exhibit 22-24% identitywith the former subunits. They possess large N-terminal, extracellularglutamate-binding domains that are homologous to the periplasmicglutamine and glutamate receptors of ABC-type uptake permeases ofGram-negative bacteria. All known members of the GIC family are fromanimals. The different channel (receptor) types exhibit distinct ionselectivities and conductance properties. The NMDA-selective largeconductance channels are highly permeable to monovalent cations andCa²⁺. The AMPA- and kainate-selective ion channels are permeableprimarily to monovalent cations with only low permeability to Ca²⁺.

The Chloride Channel (ClC) Family

[0034] The ClC family is a large family consisting of dozens ofsequenced proteins derived from Gram-negative and Gram-positivebacteria, cyanobacteria, archaea, yeast, plants and animals (Steinmeyer,K., et al., (1991), Nature 354: 301-304; Uchida, S., et al., (1993), J.Biol. Chem. 268: 3821-3824; Huang, M.-E., et al., (1994), J. Mol. Biol.242: 595-598; Kawasaki, M., et al, (1994), Neuron 12: 597-604; Fisher,W. E., et al., (1995), Genomics. 29:598-606; and Foskett, J. K. (1998),Annu. Rev. Physiol. 60: 689-717). These proteins are essentiallyubiquitous, although they are not encoded within genomes of Haemophilusinfluenzae, Mycoplasma genitalium, and Mycoplasma pneumoniae. Sequencedproteins vary in size from 395 amino acyl residues (M. jannaschii) to988 residues (man). Several organisms contain multiple ClC familyparalogues. For example, Synechocystis has two paralogues, one of 451residues in length and the other of 899 residues. Arabidopsis thalianahas at least four sequenced paralogues, (775-792 residues), humans alsohave at least five paralogues (820-988 residues), and C. elegans alsohas at least five (810-950 residues). There are nine known members inmammals, and mutations in three of the corresponding genes cause humandiseases. E. coli, Methanococcus jannaschii and Saccharomyces cerevisiaeonly have one ClC family member each. With the exception of the largerSynechocystis paralogue, all bacterial proteins are small (395-492residues) while all eukaryotic proteins are larger (687-988 residues).These proteins exhibit 10-12 putative transmembrane a-helical spanners(TMSs) and appear to be present in the membrane as homodimers. While onemember of the family, Torpedo ClC-O, has been reported to have twochannels, one per subunit, others are believed to have just one.

[0035] All functionally characterized members of the ClC familytransport chloride, some in a voltage-regulated process. These channelsserve a variety of physiological functions (cell volume regulation;membrane potential stabilization; signal transduction; transepithelialtransport, etc.). Different homologues in humans exhibit differing anionselectivities, i.e., ClC4 and ClC5 share a NO₃ ⁻>Cl⁻>Br⁻>I⁻ conductancesequence, while ClC3 has an I⁻>Cl⁻ selectivity. The ClC4 and ClC5channels and others exhibit outward rectifying currents with currentsonly at voltages more positive than +20 mV.

Animal Inward Rectifier K⁺ Channel (IRK-C) Family

[0036] IRK channels possess the “minimal channel-forming structure” withonly a P domain, characteristic of the channel proteins of the VICfamily, and two flanking transmembrane spanners (Shuck, M. E., et al.,(1994), J. Biol. Chem. 269: 24261-24270; Ashen, M. D., et al., (1995),Am. J. Physiol. 268: H506-H511; Salkoff, L. and T. Jegla (1995), Neuron15: 489-492; Aguilar-Bryan, L., et al., (1998), Physiol. Rev. 78:227-245; Ruknudin, A., et al., (1998), J. Biol. Chem. 273: 14165-14171).They may exist in the membrane as homo- or heterooligomers. They have agreater tendency to let K⁺ flow into the cell than out.Voltage-dependence may be regulated by external K⁺, by internal Mg²⁺, byinternal ATP and/or by G-proteins. The P domains of IRK channels exhibitlimited sequence similarity to those of the VIC family, but thissequence similarity is insufficient to establish homology. Inwardrectifiers play a role in setting cellular membrane potentials, and theclosing of these channels upon depolarization permits the occurrence oflong duration action potentials with a plateau phase. Inward rectifierslack the intrinsic voltage sensing helices found in VIC family channels.In a few cases, those of Kir1.1a and Kir6.2, for example, directinteraction with a member of the ABC superfamily has been proposed toconfer unique functional and regulatory properties to the heteromericcomplex, including sensitivity to ATP. The SUR1 sulfonylurea receptor(spQ09428) is the ABC protein that regulates the Kir6.2 channel inresponse to ATP, and CFTR may regulate Kir1.1a. Mutations in SUR1 arethe cause of familial persistent hyperinsulinemic hypoglycemia ininfancy (PHHI), an autosomal recessive disorder characterized byunregulated insulin secretion in the pancreas.

ATP-Gated Cation Channel (ACC) Family

[0037] Members of the ACC family (also called P2X receptors) respond toATP, a functional neurotransmitter released by exocytosis from manytypes of neurons (North, R. A. (1996), Curr. Opin. Cell Biol. 8:474-483; Soto, F., M. Garcia-Guzman and W. Stühmer (1997), J. Membr.Biol. 160: 91-100). They have been placed into seven groups (P2X₁-P2X₇)based on their pharmacological properties. These channels, whichfunction at neuron-neuron and neuron-smooth muscle junctions, may playroles in the control of blood pressure and pain sensation. They may alsofunction in lymphocyte and platelet physiology. They are found only inanimals.

[0038] The proteins of the ACC family are quite similar in sequence(>35% identity), but they possess 380-1000 amino acyl residues persubunit with variability in length localized primarily to the C-terminaldomains. They possess two transmembrane spanners, one about 30-50residues from their N-termini, the other near residues 320-340. Theextracellular receptor domains between these two spanners (of about 270residues) are well conserved with numerous conserved glycyl and cysteylresidues. The hydrophilic C-termini vary in length from 25 to 240residues. They resemble the topologically siinilar epithelial Na⁺channel (ENaC) proteins in possessing (a) N- and C-termini localizedintracellularly, (b) two putative transmembrane spanners, (c) a largeextracellular loop domain, and (d) many conserved extracellular cysteylresidues. ACC family members are, however, not demonstrably homologouswith them. ACC channels are probably hetero- or homomultimers andtransport small monovalent cations (Me+). Some also transport Ca²⁺; afew also transport small metabolites.

The Ryanodine-Inositol 1,4,5-triphosphate Receptor Ca²⁺ Channel(RIR-CaC) Family

[0039] Ryanodine (Ry)-sensitive and inositol 1,4,5-triphosphate(IP3)-sensitive Ca²⁺-release channels function in the release of Ca²⁺from intracellular storage sites in animal cells and thereby regulatevarious Ca²⁺-dependent physiological processes (Hasan, G. et al., (1992)Development 116: 967-975; Michikawa, T., et al., (1994), J. Biol. Chem.269: 9184-9189; Tunwell, R. E. A., (1996), Biochem. J. 318: 477-487;Lee, A. G. (1996) Biomembranes, Vol. 6, Transmembrane Receptors andChannels (A. G. Lee, ed.), JAI Press, Denver, Colo., pp 291-326;Mikoshiba, K., et al., (1996) J. Biochem. Biomem. 6: 273-289). Ryreceptors occur primarily in muscle cell sarcoplasmic reticular (SR)membranes, and IP3 receptors occur primarily in brain cell endoplasmicreticular (ER) membranes where they effect release of Ca²⁺ into thecytoplasm upon activation (opening) of the channel.

[0040] The Ry receptors are activated as a result of the activity ofdihydropyridine-sensitive Ca²⁺ channels. The latter are members of thevoltage-sensitive ion channel (VIC) family. Dihydropyridine-sensitivechannels are present in the T-tubular systems of muscle tissues.

[0041] Ry receptors are homotetrameric complexes with each subunitexhibiting a molecular size of over 500,000 daltons (about 5,000 aminoacyl residues). They possess C-terminal domains with six putativetransmembrane a -helical spanners (TMSs). Putative pore-formingsequences occur between the fifth and sixth TMSs as suggested formembers of the VIC family. The large N-terminal hydrophilic domains andthe small C-terminal hydrophilic domains are localized to the cytoplasm.Low resolution 3-dimensional structural data are available. Mammalspossess at least three isoforms that probably arose by gene duplicationand divergence before divergence of the mammalian species. Homologuesare present in humans and Caenorabditis elegans.

[0042] IP₃ receptors resemble Ry receptors in many respects. (1) Theyare homotetrameric complexes with each subunit exhibiting a molecularsize of over 300,000 daltons (about 2,700 amino acyl residues). (2) Theypossess C-terminal channel domains that are homologous to those of theRy receptors. (3) The channel domains possess six putative TMSs and aputative channel lining region between TMSs 5 and 6. (4) Both the largeN-terminal domains and the smaller C-terminal tails face the cytoplasm.(5) They possess covalently linked carbohydrate on extracytoplasmicloops of the channel domains. (6) They have three currently recognizedisoforms (types 1, 2, and 3) in mammals which are subject todifferential regulation and have different tissue distributions.

[0043] IP3 receptors possess three domains: N-terminal IP₃-bindingdomains, central coupling or regulatory domains and C-terminal channeldomains. Channels are activated by IP₃ binding, and like the Ryreceptors, the activities of the IP3 receptor channels are regulated byphosphorylation of the regulatory domains, catalyzed by various proteinkinases. They predominate in the endoplasmic reticular membranes ofvarious cell types in the brain but have also been found in the plasmamembranes of some nerve cells derived from a variety of tissues.

[0044] The channel domains of the Ry and IP₃ receptors comprise acoherent family that in spite of apparent structural similarities, donot show appreciable sequence similarity of the proteins of the VICfamily. The Ry receptors and the IP₃ receptors cluster separately on theRIR-CaC family tree. They both have homologues in Drosophila. Based onthe phylogenetic tree for the family, the family probably evolved in thefollowing sequence: (1) A gene duplication event occurred that gave riseto Ry and IP₃ receptors in invertebrates. (2) Vertebrates evolved frominvertebrates. (3) The three isoforms of each receptor arose as a resultof two distinct gene duplication events. (4) These isoforms weretransmitted to mammals before divergence of the mammalian species.

The Organellar Chloride Channel (O-ClC) Family

[0045] Proteins of the O-ClC family are voltage-sensitive chloridechannels found in intracellular membranes but not the plasma membranesof animal cells (Landry, D, et al., (1993), J. Biol. Chem. 268:14948-14955; Valenzuela, Set al., (1997), J. Biol. Chem. 272:12575-12582; and Duncan, R. R., et al., (1997), J. Biol. Chem. 272:23880-23886).

[0046] They are found in human nuclear membranes, and the bovine proteintargets to the microsomes, but not the plasma membrane, when expressedin Xenopus laevis oocytes. These proteins are thought to function in theregulation of the membrane potential and in transepithelial ionabsorption and secretion in the kidney. They possess two putativetransmembrane a-helical spanners (TMSs) with cytoplasmic N- andC-termini and a large luminal loop that may be glycosylated. The bovineprotein is 437 amino acyl residues in length and has the two putativeTMSs at positions 223-239 and 367-385. The human nuclear protein is muchsmaller (241 residues). A C. elegans homologue is 260 residues long.

Vacuolar ATPase

[0047] The novel human protein provided by the present invention isrelated to the family of vacuolar ATPases (V-ATPases), which are alsoknown by such names as vacuolar ATP synthases and vacuolar protonATPases; specifically, the protein of the present invention is aV-ATPase subunit. V-ATPases translocate protons into intracellularorganelles or across plasma membranes of particular cells such asosteoclasts and renal intercalated cells (van Hille et al., BiochemBiophys Res Commun 1993 November 30;197(1):15-21). V-ATPase is alsoimportant for acidifying intracellular compartments in eukaryotic cells.V-ATPase comprises at least 9 subunits, including six catalytic subunitsfor binding and hydrolyzing ATP and three regulatory subunits (van Hilleet al., Biochem Biophys Res Commun 1993 November 30;197(1):15-21).

[0048] Physophilin, an oligomeric protein, and synaptophysin, a synapticvesicle protein, may form a exocytotic fusion pore. Physophilin peptidesconstitute the Ac39 subunit of V-ATPase. Ac39 is part of a synaptosomalcomplex that includes synaptophysin, synaptobrevin II, and subunits cand Ac115 of the V0 sector of V-ATPase. The amino acid sequence ofphysophilin matches that of Ac39. Transcription of V-ATPase andsynaptophysin genes may be coordinately controlled. Ac39/physophilin mayinactivate V-ATPase by disassembly of the V1 sector of V-ATPase(Carrion-Vazquez et al., Eur J Neurosci 1998 March;10(3):1153-66).

[0049] For a further review of V-ATPases, see Bauerle et al., J BiolChem 1993 June 15;268(17):12749-57; Wilms et al, J Biol Chem 1996 August2;271(31):18843-52; Takase et al., J Biol Chem 1994 April15;269(15):11037-44; Wang et al., J Biol Chem 1988 November25;263(33):17638-42; and Merzendorfer et al, FEBS Lett 1997 July14;411(2-3): 239-44.

[0050] Transporter proteins, particularly members of the vacuolar ATPasesubfamily, are a major target for drug action and development.Accordingly, it is valuable to the field of pharmaceutical developmentto identify and characterize previously unknown transport proteins. Thepresent invention advances the state of the art by providing previouslyunidentified human transport proteins.

SUMMARY OF THE INVENTION

[0051] The present invention is based in part on the identification ofamino acid sequences of human transporter peptides and proteins that arerelated to the vacuolar ATPase transporter subfamily, as well as allelicvariants and other mammalian orthologs thereof. These unique peptidesequences, and nucleic acid sequences that encode these peptides, can beused as models for the development of human therapeutic targets, aid inthe identification of therapeutic proteins, and serve as targets for thedevelopment of human therapeutic agents that modulate transporteractivity in cells and tissues that express the transporter. Experimentaldata as provided in FIG. 1 indicates expression in humans in the kidneyand lung, as well as in kidney tumors.

DESCRIPTION OF THE FIGURE SHEETS

[0052]FIG. 1 provides the nucleotide sequence of a cDNA molecule thatencodes the transporter protein of the present invention. (SEQ ID NO: 1)In addition structure and functional information is provided, such asATG start, stop and tissue distribution, where available, that allowsone to readily determine specific uses of inventions based on thismolecular sequence. Experimental data as provided in FIG. 1 indicatesexpression in humans in the kidney and lung, as well as in kidneytumors.

[0053]FIG. 2 provides the predicted amino acid sequence of thetransporter of the present invention. (SEQ ID NO: 2) In additionstructure and functional information such as protein family, function,and modification sites is provided where available, allowing one toreadily determine specific uses of inventions based on this molecularsequence.

[0054]FIG. 3 provides genomic sequences that span the gene encoding thetransporter protein of the present invention. (SEQ ID NO: 3) In additionstructure and functional information, such as intron/exon structure,promoter location, etc., is provided where available, allowing one toreadily determine specific uses of inventions based on this molecularsequence. As illustrated in FIG. 3, SNPs were identified at 77 differentnucleotide positions.

DETAILED DESCRIPTION OF THE INVENTION General Description

[0055] The present invention is based on the sequencing of the humangenome. During the sequencing and assembly of the human genome, analysisof the sequence information revealed previously unidentified fragmentsof the human genome that encode peptides that share structural and/orsequence homology to protein/peptide/domains identified andcharacterized within the art as being a transporter protein or part of atransporter protein and are related to the vacuolar ATPase transportersubfamily. Utilizing these sequences, additional genomic sequences wereassembled and transcript and/or cDNA sequences were isolated andcharacterized. Based on this analysis, the present invention providesamino acid sequences of human transporter peptides and proteins that arerelated to the vacuolar ATPase transporter subfamily, nucleic acidsequences in the form of transcript sequences, cDNA sequences and/orgenomic sequences that encode these transporter peptides and proteins,nucleic acid variation (allelic information), tissue distribution ofexpression, and information about the closest art knownprotein/peptide/domain that has structural or sequence homology to thetransporter of the present invention.

[0056] In addition to being previously unknown, the peptides that areprovided in the present invention are selected based on their ability tobe used for the development of commercially important products andservices. Specifically, the present peptides are selected based onhomology and/or structural relatedness to known transporter proteins ofthe vacuolar ATPase transporter subfamily and the expression patternobserved. Experimental data as provided in FIG. 1 indicates expressionin humans in the kidney and lung, as well as in kidney tumors. The arthas clearly established the commercial importance of members of thisfamily of proteins and proteins that have expression patterns similar tothat of the present gene. Some of the more specific features of thepeptides of the present invention, and the uses thereof, are describedherein, particularly in the Background of the Invention and in theannotation provided in the Figures, and/or are known within the art foreach of the known vacuolar ATPase family or subfamily of transporterproteins.

Specific Embodiments

[0057] Peptide Molecules

[0058] The present invention provides nucleic acid sequences that encodeprotein molecules that have been identified as being members of thetransporter family of proteins and are related to the vacuolar ATPasetransporter subfamily (protein sequences are provided in FIG. 2,transcript/cDNA sequences are provided in FIGS. 1 and sequences areprovided in FIG. 3). The peptide sequences provided in FIG. 2, as wellas the obvious variants described herein, particularly allelic variantsas identified herein and using the information in FIG. 3, will bereferred herein as the transporter peptides of the present invention,transporter peptides, or peptides/proteins of the present invention.

[0059] The present invention provides isolated peptide and proteinmolecules that consist of, consist essentially of, or comprising theamino acid sequences of the transporter peptides disclosed in the FIG.2, (encoded by the nucleic acid molecule shown in FIG. 1,transcript/cDNA or FIG. 3, genomic sequence), as well as all obviousvariants of these peptides that are within the art to make and use. Someof these variants are described in detail below.

[0060] As used herein, a peptide is said to be “isolated” or “purified”when it is substantially free of cellular material or free of chemicalprecursors or other chemicals. The peptides of the present invention canbe purified to homogeneity or other degrees of purity. The level ofpurification will be based on the intended use. The critical feature isthat the preparation allows for the desired function of the peptide,even if in the presence of considerable amounts of other components (thefeatures of an isolated nucleic acid molecule is discussed below).

[0061] In some uses, “substantially free of cellular material” includespreparations of the peptide having less than about 30% (by dry weight)other proteins (i.e., contaminating protein), less than about 20% otherproteins, less than about 10% other proteins, or less than about 5%other proteins. When the peptide is recombinantly produced, it can alsobe substantially free of culture medium, i.e., culture medium representsless than about 20% of the volume of the protein preparation.

[0062] The language “substantially free of chemical precursors or otherchemicals” includes preparations of the peptide in which it is separatedfrom chemical precursors or other chemicals that are involved in itssynthesis. In one embodiment, the language “substantially free ofchemical precursors or other chemicals” includes preparations of thetransporter peptide having less than about 30% (by dry weight) chemicalprecursors or other chermicals, less than about 20% chemical precursorsor other chemicals, less than about 10% chemical precursors or otherchemicals, or less than about 5% chemical precursors or other chemicals.

[0063] The isolated transporter peptide can be purified from cells thatnaturally express it, purified from cells that have been altered toexpress it (recombinant), or synthesized using known protein synthesismethods. Experimental data as provided in FIG. 1 indicates expression inhumans in the kidney and lung, as well as in kidney tumors. For example,a nucleic acid molecule encoding the transporter peptide is cloned intoan expression vector, the expression vector introduced into a host celland the protein expressed in the host cell. The protein can then beisolated from the cells by an appropriate purification scheme usingstandard protein purification techniques. Many of these techniques aredescribed in detail below.

[0064] Accordingly, the present invention provides proteins that consistof the amino acid sequences provided in FIG. 2 (SEQ ID NO: 2), forexample, proteins encoded by the transcript/cDNA nucleic acid sequencesshown in FIG. 1 (SEQ ID NO: 1) and the genomic sequences provided inFIG. 3 (SEQ ID NO: 3). The amino acid sequence of such a protein isprovided in FIG. 2. A protein consists of an amino acid sequence whenthe amino acid sequence is the final amino acid sequence of the protein.

[0065] The present invention further provides proteins that consistessentially of the amino acid sequences provided in FIG. 2 (SEQ ID NO:2), for example, proteins encoded by the transcript/cDNA nucleic acidsequences shown in FIG. 1 (SEQ ID NO: 1) and the genomic sequencesprovided in FIG. 3 (SEQ ID NO: 3). A protein consists essentially of anamino acid sequence when such an amino acid sequence is present withonly a few additional amino acid residues, for example from about 1 toabout 100 or so additional residues, typically from 1 to about 20additional residues in the final protein.

[0066] The present invention further provides proteins that comprise theamino acid sequences provided in FIG. 2 (SEQ ID NO: 2), for example,proteins encoded by the transcript/cDNA nucleic acid sequences shown inFIG. 1 (SEQ ID NO: 1) and the genomic sequences provided in FIG. 3 (SEQID NO: 3). A protein comprises an amino acid sequence when the aminoacid sequence is at least part of the final amino acid sequence of theprotein. In such a fashion, the protein can be only the peptide or haveadditional amino acid molecules, such as amino acid residues (contiguousencoded sequence) that are naturally associated with it or heterologousamino acid residues/peptide sequences. Such a protein can have a fewadditional amino acid residues or can comprise several hundred or moreadditional amino acids. The preferred classes of proteins that arecomprised of the transporter peptides of the present invention are thenaturally occurring mature proteins. A brief description of how varioustypes of these proteins can be made/isolated is provided below.

[0067] The transporter peptides of the present invention can be attachedto heterologous sequences to form chimeric or fusion proteins. Suchchimeric and fusion proteins comprise a transporter peptide operativelylinked to a heterologous protein having an amino acid sequence notsubstantially homologous to the transporter peptide. “Operativelylinked” indicates that the transporter peptide and the heterologousprotein are fused in-frame. The heterologous protein can be fused to theN-terminus or C-terminus of the transporter peptide.

[0068] In some uses, the fusion protein does not affect the activity ofthe transporter peptide per se. For example, the fusion protein caninclude, but is not limited to, enzymatic fusion proteins, for examplebeta-galactosidase fusions, yeast two-hybrid GAL fusions, poly-Hisfusions, MYC-tagged, HI-tagged and Ig fusions. Such fusion proteins,particularly poly-His fusions, can facilitate the purification ofrecombinant transporter peptide. In certain host cells (e.g., mammalianhost cells), expression and/or secretion of a protein can be increasedby using a heterologous signal sequence.

[0069] A chimeric or fusion protein can be produced by standardrecombinant DNA techniques. For example, DNA fragments coding for thedifferent protein sequences are ligated together in-frame in accordancewith conventional techniques. In another embodiment, the fusion gene canbe synthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers which give rise to complementaryoverhangs between two consecutive gene fragments which can subsequentlybe annealed and re-amplified to generate a chimeric gene sequence (seeAusubel et al., Current Protocols in Molecular Biology, 1992). Moreover,many expression vectors are commercially available that already encode afusion moiety (e.g., a GST protein). A transporter peptide-encodingnucleic acid can be cloned into such an expression vector such that thefusion moiety is linked in-frame to the transporter peptide.

[0070] As mentioned above, the present invention also provides andenables obvious variants of the amino acid sequence of the proteins ofthe present invention, such as naturally occurring mature forms of thepeptide, allelic/sequence variants of the peptides, non-naturallyoccurring recombinantly derived variants of the peptides, and orthologsand paralogs of the peptides. Such variants can readily be generatedusing art-known techniques in the fields of recombinant nucleic acidtechnology and protein biochemistry. It is understood, however, thatvariants exclude any amino acid sequences disclosed prior to theinvention.

[0071] Such variants can readily be identified/made using moleculartechniques and the sequence information disclosed herein. Further, suchvariants can readily be distinguished from other peptides based onsequence and/or structural homology to the transporter peptides of thepresent invention. The degree of homology/identity present will be basedprimarily on whether the peptide is a functional variant ornon-functional variant, the amount of divergence present in the paralogfamily and the evolutionary distance between the orthologs.

[0072] To determine the percent identity of two amino acid sequences ortwo nucleic acid sequences, the sequences are aligned for optimalcomparison purposes (e.g., gaps can be introduced in one or both of afirst and a second amino acid or nucleic acid sequence for optimalalignment and non-homologous sequences can be disregarded for comparisonpurposes). In a preferred embodiment, at least 30%, 40%, 50%, 60%, 70%,80%, or 90% or more of a reference sequence is aligned for comparisonpurposes. The amino acid residues or nucleotides at corresponding aminoacid positions or nucleotide positions are then compared. When aposition in the first sequence is occupied by the same amino acidresidue or nucleotide as the corresponding position in the secondsequence, then the molecules are identical at that position (as usedherein amino acid or nucleic acid “identity” is equivalent to amino acidor nucleic acid “homology”). The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences.

[0073] The comparison of sequences and determination of percent identityand similarity between two sequences can be accomplished using amathematical algorithm. (Computational Molecular Biology, Lesk, A. M.,ed., Oxford University Press, New York, 1988; Biocomputing: Informaticsand Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis ofsequence Data, Part 1, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991). In a preferred embodiment, the percent identity betweentwo amino acid sequences is determined using the Needleman and Wunsch(J. Mol. Biol. (48):444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat http://www.gcg.com), using either a Blossom 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, thepercent identity between two nucleotide sequences is determined usingthe GAP program in the GCG software package (Devereux, J., et al.,Nucleic Acids Res. 12(1):387 (1984)) (available at http://www.gcg.com),using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, thepercent identity between two amino acid or nucleotide sequences isdetermined using the algorithm of E. Myers and W. Miller (CABIOS,4:11-17 (1989)) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4.

[0074] The nucleic acid and protein sequences of the present inventioncan further be used as a “query sequence” to perform a search againstsequence databases to, for example, identify other family members orrelated sequences. Such searches can be performed using the NBLAST andXBLAST programs (version 2.0) of Altschul, et al. (J Mol. Biol.215:403-10 (1990)). BLAST nucleotide searches can be performed with theNBLAST program, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to the nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to the proteinsof the invention. To obtain gapped alignments for comparison purposes,Gapped BLAST can be utilized as described in Altschul et al. (NucleicAcids Res. 25(17):3389-3402 (1997)). When utilizing BLAST and gappedBLAST programs, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used.

[0075] Full-length pre-processed forms, as well as mature processedforms, of proteins that comprise one of the peptides of the presentinvention can readily be identified as having complete sequence identityto one of the transporter peptides of the present invention as well asbeing encoded by the same genetic locus as the transporter peptideprovided herein. The gene encoding the novel transporter protein of thepresent invention is located on a genome component that has been mappedto human chromosome 8 (as indicated in FIG. 3), which is supported bymultiple lines of evidence, such as STS and BAC map data.

[0076] Allelic variants of a transporter peptide can readily beidentified as being a human protein having a high degree (significant)of sequence homology/identity to at least a portion of the transporterpeptide as well as being encoded by the same genetic locus as thetransporter peptide provided herein. Genetic locus can readily bedetermined based on the genomic information provided in FIG. 3, such asthe genormic sequence mapped to the reference human. The gene encodingthe novel transporter protein of the present invention is located on agenome component that has been mapped to human chromosome 8 (asindicated in FIG. 3), which is supported by multiple lines of evidence,such as STS and BAC map data. As used herein, two proteins (or a regionof the proteins) have significant homology when the amino acid sequencesare typically at least about 70-80%, 80-90%, and more typically at leastabout 90-95% or more homologous. A significantly homologous amino acidsequence, according to the present invention, will be encoded by anucleic acid sequence that will hybridize to a transporter peptideencoding nucleic acid molecule under stringent conditions as more fullydescribed below.

[0077]FIG. 3 provides information on SNPs that have been found in thegene encoding the transporter protein of the present invention. SNPswere identified at 77 different nucleotide positions. These SNPs,particularly the SNPs located in the first intron, may affectcontrol/regulatory elements.

[0078] Paralogs of a transporter peptide can readily be identified ashaving some degree of significant sequence homology/identity to at leasta portion of the transporter peptide, as being encoded by a gene fromhumans, and as having similar activity or function. Two proteins willtypically be considered paralogs when the amino acid sequences aretypically at least about 60% or greater, and more typically at leastabout 70% or greater homology through a given region or domain. Suchparalogs will be encoded by a nucleic acid sequence that will hybridizeto a transporter peptide encoding nucleic acid molecule under moderateto stringent conditions as more fully described below.

[0079] Orthologs of a transporter peptide can readily be identified ashaving some degree of significant sequence homology/identity to at leasta portion of the transporter peptide as well as being encoded by a genefrom another organism. Preferred orthologs will be isolated frommammals, preferably primates, for the development of human therapeutictargets and agents. Such orthologs will be encoded by a nucleic acidsequence that will hybridize to a transporter peptide encoding nucleicacid molecule under moderate to stringent conditions, as more fullydescribed below, depending on the degree of relatedness of the twoorganisms yielding the proteins.

[0080] Non-naturally occurring variants of the transporter peptides ofthe present invention can readily be generated using recombinanttechniques. Such variants include, but are not limited to deletions,additions and substitutions in the amino acid sequence of thetransporter peptide. For example, one class of substitutions areconserved amino acid substitution. Such substitutions are those thatsubstitute a given amino acid in a transporter peptide by another aminoacid of like characteristics. Typically seen as conservativesubstitutions are the replacements, one for another, among the aliphaticamino acids Ala, Val, Leu, and Ile; interchange of the hydroxyl residuesSer and Thr; exchange of the acidic residues Asp and Glu; substitutionbetween the amide residues Asn and Gln; exchange of the basic residuesLys and Arg; and replacements among the aromatic residues Phe and Tyr.Guidance concerning which amino acid changes are likely to bephenotypically silent are found in Bowie et al., Science 247:1306-1310(1990).

[0081] Variant transporter peptides can be fully functional or can lackfunction in one or more activities, e.g. ability to bind ligand, abilityto transport ligand, ability to mediate signaling, etc. Fully functionalvariants typically contain only conservative variation or variation innon-critical residues or in non-critical regions. FIG. 2 provides theresult of protein analysis and can be used to identify criticaldomains/regions. Functional variants can also contain substitution ofsimilar amino acids that result in no change or an insignificant changein function. Alternatively, such substitutions may positively ornegatively affect function to some degree.

[0082] Non-functional variants typically contain one or morenon-conservative amino acid substitutions, deletions, insertions,inversions, or truncation or a substitution, insertion, inversion, ordeletion in a critical residue or critical region.

[0083] Amino acids that are essential for function can be identified bymethods known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham et al., Science 244:1081-1085(1989)), particularly using the results provided in FIG. 2. The latterprocedure introduces single alanine mutations at every residue in themolecule. The resulting mutant molecules are then tested for biologicalactivity such as transporter activity or in assays such as an in vitroproliferative activity. Sites that are critical for bindingpartner/substrate binding can also be determined by structural analysissuch as crystallization, nuclear magnetic resonance or photoaffinitylabeling (Smith et al., J. Mol. Biol. 224:899-904 (1992); de Vos et al.Science 255:306-312 (1992)).

[0084] The present invention further provides fragments of thetransporter peptides, in addition to proteins and peptides that compriseand consist of such fragments, particularly those comprising theresidues identified in FIG. 2. The fragments to which the inventionpertains, however, are not to be construed as encompassing fragmentsthat may be disclosed publicly prior to the present invention.

[0085] As used herein, a fragment comprises at least 8, 10, 12, 14, 16,or more contiguous amino acid residues from a transporter peptide. Suchfragments can be chosen based on the ability to retain one or more ofthe biological activities of the transporter peptide or could be chosenfor the ability to perform a function, e.g. bind a substrate or act asan immunogen. Particularly important fragments are biologically activefragments, peptides that are, for example, about 8 or more amino acidsin length. Such fragments will typically comprise a domain or motif ofthe transporter peptide, e.g., active site, a transmembrane domain or asubstrate-binding domain. Further, possible fragments include, but arenot limited to, domain or motif containing fragments, soluble peptidefragments, and fragments containing immunogenic structures. Predicteddomains and functional sites are readily identifiable by computerprograms well known and readily available to those of skill in the art(e.g., PROSITE analysis). The results of one such analysis are providedin FIG. 2.

[0086] Polypeptides often contain amino acids other than the 20 aminoacids commonly referred to as the 20 naturally occurring amino acids.Further, many amino acids, including the terminal amino acids, may bemodified by natural processes, such as processing and otherpost-translational modifications, or by chemical modification techniqueswell known in the art. Common modifications that occur naturally intransporter peptides are described in basic texts, detailed monographs,and the research literature, and they are well known to those of skillin the art (some of these features are identified in FIG. 2).

[0087] Known modifications include, but are not limited to, acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent crosslinks, formation of cystine, formation ofpyroglutamate, formylation, gamma carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination.

[0088] Such modifications are well known to those of skill in the artand have been described in great detail in the scientific literature.Several particularly comnmon modifications, glycosylation, lipidattachment, sulfation, gamma-carboxylation of glutamic acid residues,hydroxylation and ADP-ribosylation, for instance, are described in mostbasic texts, such as Proteins—Structure and Molecular Properties, 2ndEd., T. E. Creighton, W. H. Freeman and Company, New York (1993). Manydetailed reviews are available on this subject, such as by Wold, F.,Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed.,Academic Press, New York 1-12 (1983); Seifter et al. (Meth. Enzymol.182: 626-646 (1990)) and Rattan et al. (Ann. N. Y. Acad. Sci. 663:48-62(1992)).

[0089] Accordingly, the transporter peptides of the present inventionalso encompass derivatives or analogs in which a substituted amino acidresidue is not one encoded by the genetic code, in which a substituentgroup is included, in which the mature transporter peptide is fused withanother compound, such as a compound to increase the half-life of thetransporter peptide (for example, polyethylene glycol), or in which theadditional amino acids are fused to the mature transporter peptide, suchas a leader or secretory sequence or a sequence for purification of themature transporter peptide or a pro-protein sequence.

[0090] Protein/Peptide Uses

[0091] The proteins of the present invention can be used in substantialand specific assays related to the functional information provided inthe Figures; to raise antibodies or to elicit another immune response;as a reagent (including the labeled reagent) in assays designed toquantitatively determine levels of the protein (or its binding partneror ligand) in biological fluids; and as markers for tissues in which thecorresponding protein is preferentially expressed (either constitutivelyor at a particular stage of tissue differentiation or development or ina disease state). Where the protein binds or potentially binds toanother protein or ligand (such as, for example, in atransporter-effector protein interaction or transporter-ligandinteraction), the protein can be used to identify the bindingpartner/ligand so as to develop a system to identify inhibitors of thebinding interaction. Any or all of these uses are capable of beingdeveloped into reagent grade or kit format for commercialization ascommercial products.

[0092] Methods for performing the uses listed above are well known tothose skilled in the art. References disclosing such methods include“Molecular Cloning: A Laboratory Manual”, 2d ed., Cold Spring HarborLaboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds.,1989, and “Methods in Enzymology: Guide to Molecular CloningTechniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.

[0093] The potential uses of the peptides of the present invention arebased primarily on the source of the protein as well as the class/actionof the protein. For example, transporters isolated from humans and theirhuman/mammalian orthologs serve as targets for identifying agents foruse in mammalian therapeutic applications, e.g. a human drug,particularly in modulating a biological or pathological response in acell or tissue that expresses the transporter. Experimental data asprovided in FIG. 1 indicates that the transporter proteins of thepresent invention are expressed in humans in both normal kidney tissueand kidney tumors, as indicated by virtual northern blot analysis. Inaddition, PCR-based tissue screening panels indicate expression in thehuman lung. A large percentage of pharmaceutical agents are beingdeveloped that modulate the activity of transporter proteins,particularly members of the vacuolar ATPase subfamily (see Background ofthe Invention). The structural and functional information provided inthe Background and Figures provide specific and substantial uses for themolecules of the present invention, particularly in combination with theexpression information provided in FIG. 1. Experimental data as providedin FIG. 1 indicates expression in humans in the kidney and lung, as wellas in kidney tumors. Such uses can readily be determined using theinformation provided herein, that known in the art and routineexperimentation.

[0094] The proteins of the present invention (including variants andfragments that may have been disclosed prior to the present invention)are useful for biological assays related to transporters that arerelated to members of the vacuolar ATPase subfamily. Such assays involveany of the known transporter functions or activities or propertiesuseful for diagnosis and treatment of transporter-related conditionsthat are specific for the subfamily of transporters that the one of thepresent invention belongs to, particularly in cells and tissues thatexpress the transporter. Experimental data as provided in FIG. 1indicates that the transporter proteins of the present invention areexpressed in humans in both normal kidney tissue and kidney tumors, asindicated by virtual northern blot analysis. In addition, PCR-basedtissue screening panels indicate expression in the human lung. Theproteins of the present invention are also useful in drug screeningassays, in cell-based or cell-free systems ((Hodgson, Bio/technology,1992, September 10(9);973-80). Cell-based systems can be native, i.e.,cells that normally express the transporter, as a biopsy or expanded incell culture. Experimental data as provided in FIG. 1 indicatesexpression in humans in the kidney and lung, as well as in kidneytumors. In an alternate embodiment, cell-based assays involverecombinant host cells expressing the transporter protein.

[0095] The polypeptides can be used to identify compounds that modulatetransporter activity of the protein in its natural state or an alteredform that causes a specific disease or pathology associated with thetransporter. Both the transporters of the present invention andappropriate variants and fragments can be used in high-throughputscreens to assay candidate compounds for the ability to bind to thetransporter. These compounds can be further screened against afunctional transporter to determine the effect of the compound on thetransporter activity. Further, these compounds can be tested in animalor invertebrate systems to determine activity/effectiveness. Compoundscan be identified that activate (agonist) or inactivate (antagonist) thetransporter to a desired degree.

[0096] Further, the proteins of the present invention can be used toscreen a compound for the ability to stimulate or inhibit interactionbetween the transporter protein and a molecule that normally interactswith the transporter protein, e.g. a substrate or a component of thesignal pathway that the transporter protein normally interacts (forexample, another transporter). Such assays typically include the stepsof combining the transporter protein with a candidate compound underconditions that allow the transporter protein, or fragment, to interactwith the target molecule, and to detect the formation of a complexbetween the protein and the target or to detect the biochemicalconsequence of the interaction with the transporter protein and thetarget, such as any of the associated effects of signal transductionsuch as changes in membrane potential, protein phosphorylation, cAMPturnover, and adenylate cyclase activation, etc.

[0097] Candidate compounds include, for example, 1) peptides such assoluble peptides, including Ig-tailed fusion peptides and members ofrandom peptide libraries (see, e.g., Lam et al., Nature 354:82-84(1991); Houghten et al., Nature 354:84-86 (1991)) and combinatorialchemistry-derived molecular libraries made of D- and/or L- configurationamino acids; 2) phosphopeptides (e.g., members of random and partiallydegenerate, directed phosphopeptide libraries, see, e.g., Songyang etal., Cell 72:767-778 (1993)); 3) antibodies (e.g., polyclonal,monoclonal, humanized, anti-idiotypic, chimeric, and single chainantibodies as well as Fab, F(ab′)₂, Fab expression library fragments,and epitope-binding fragments of antibodies); and 4) small organic andinorganic molecules (e.g., molecules obtained from combinatorial andnatural product libraries).

[0098] One candidate compound is a soluble fragment of the receptor thatcompetes for ligand binding. Other candidate compounds include mutanttransporters or appropriate fragments containing mutations that affecttransporter function and thus compete for ligand. Accordingly, afragment that competes for ligand, for example with a higher affinity,or a fragment that binds ligand but does not allow release, isencompassed by the invention.

[0099] The invention further includes other end point assays to identifycompounds that modulate (stimulate or inhibit) transporter activity. Theassays typically involve an assay of events in the signal transductionpathway that indicate transporter activity. Thus, the transport of aligand, change in cell membrane potential, activation of a protein, achange in the expression of genes that are up- or down-regulated inresponse to the transporter protein dependent signal cascade can beassayed.

[0100] Any of the biological or biochemical functions mediated by thetransporter can be used as an endpoint assay. These include all of thebiochemical or biochemical/biological events described herein, in thereferences cited herein, incorporated by reference for these endpointassay targets, and other functions known to those of ordinary skill inthe art or that can be readily identified using the information providedin the Figures, particularly FIG. 2. Specifically, a biological functionof a cell or tissues that expresses the transporter can be assayed.Experimental data as provided in FIG. 1 indicates that the transporterproteins of the present invention are expressed in humans in both normalkidney tissue and kidney tumors, as indicated by virtual northern blotanalysis. In addition, PCR-based tissue screening panels indicateexpression in the human lung.

[0101] Binding and/or activating compounds can also be screened by usingchimeric transporter proteins in which the amino terminal extracellulardomain, or parts thereof, the entire transmembrane domain or subregions,such as any of the seven transmembrane segments or any of theintracellular or extracellular loops and the carboxy terminalintracellular domain, or parts thereof, can be replaced by heterologousdomains or subregions. For example, a ligand-binding region can be usedthat interacts with a different ligand then that which is recognized bythe native transporter. Accordingly, a different set of signaltransduction components is available as an end-point assay foractivation. This allows for assays to be performed in other than thespecific host cell from which the transporter is derived.

[0102] The proteins of the present invention are also useful incompetition binding assays in methods designed to discover compoundsthat interact with the transporter (e.g. binding partners and/orligands). Thus, a compound is exposed to a transporter polypeptide underconditions that allow the compound to bind or to otherwise interact withthe polypeptide. Soluble transporter polypeptide is also added to themixture. If the test compound interacts with the soluble transporterpolypeptide, it decreases the amount of complex formed or activity fromthe transporter target. This type of assay is particularly useful incases in which compounds are sought that interact with specific regionsof the transporter. Thus, the soluble polypeptide that competes with thetarget transporter region is designed to contain peptide sequencescorresponding to the region of interest.

[0103] To perform cell free drug screening assays, it is sometimesdesirable to immobilize either the transporter protein, or fragment, orits target molecule to facilitate separation of complexes fromuncomplexed forms of one or both of the proteins, as well as toaccommodate automation of the assay.

[0104] Techniques for immobilizing proteins on matrices can be used inthe drug screening assays. In one embodiment, a fusion protein can beprovided which adds a domain that allows the protein to be bound to amatrix. For example, glutathione-S-transferase fusion proteins can beadsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis,Mo.) or glutathione derivatized microtitre plates, which are thencombined with the cell lysates (e.g., ³⁵S-labeled) and the candidatecompound, and the mixture incubated under conditions conducive tocomplex formation (e.g., at physiological conditions for salt and pH).Following incubation, the beads are washed to remove any unbound label,and the matrix immobilized and radiolabel determined directly, or in thesupernatant after the complexes are dissociated. Alternatively, thecomplexes can be dissociated from the matrix, separated by SDS-PAGE, andthe level of transporter-binding protein found in the bead fractionquantitated from the gel using standard electrophoretic techniques. Forexample, either the polypeptide or its target molecule can beimmobilized utilizing conjugation of biotin and streptavidin usingtechniques well known in the art. Alternatively, antibodies reactivewith the protein but which do not interfere with binding of the proteinto its target molecule can be derivatized to the wells of the plate, andthe protein trapped in the wells by antibody conjugation. Preparationsof a transporter-binding protein and a candidate compound are incubatedin the transporter protein-presenting wells and the amount of complextrapped in the well can be quantitated. Methods for detecting suchcomplexes, in addition to those described above for the GST-immobilizedcomplexes, include immunodetection of complexes using antibodiesreactive with the transporter protein target molecule, or which arereactive with transporter protein and compete with the target molecule,as well as enzyme-linked assays which rely on detecting an enzymaticactivity associated with the target molecule.

[0105] Agents that modulate one of the transporters of the presentinvention can be identified using one or more of the above assays, aloneor in combination. It is generally preferable to use a cell-based orcell free system first and then confirm activity in an animal or othermodel system. Such model systems are well known in the art and canreadily be employed in this context.

[0106] Modulators of transporter protein activity identified accordingto these drug screening assays can be used to treat a subject with adisorder mediated by the transporter pathway, by treating cells ortissues that express the transporter. Experimental data as provided inFIG. 1 indicates expression in humans in the kidney and lung, as well asin kidney tumors. These methods of treatment include the steps ofadministering a modulator of transporter activity in a pharmaceuticalcomposition to a subject in need of such treatment, the modulator beingidentified as described herein.

[0107] In yet another aspect of the invention, the transporter proteinscan be used as “bait proteins” in a two-hybrid assay or three-hybridassay (see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartelet al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene8:1693-1696; and Brent W094/10300), to identify other proteins, whichbind to or interact with the transporter and are involved in transporteractivity. Such transporter-binding proteins are also likely to beinvolved in the propagation of signals by the transporter proteins ortransporter targets as, for example, downstream elements of atransporter-mediated signaling pathway. Alternatively, suchtransporter-binding proteins are likely to be transporter inhibitors.

[0108] The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for a transporterprotein is fused to a gene encoding the DNA binding domain of a knowntranscription factor (e.g., GAL-4). In the other construct, a DNAsequence, from a library of DNA sequences, that encodes an unidentifiedprotein (“prey” or “sample”) is fused to a gene that codes for theactivation domain of the known transcription factor. If the “bait” andthe “prey” proteins are able to interact, in vivo, forming atransporter-dependent complex, the DNA-binding and activation domains ofthe transcription factor are brought into close proximity. Thisproximity allows transcription of a reporter gene (e.g., LacZ) which isoperably linked to a transcriptional regulatory site responsive to thetranscription factor. Expression of the reporter gene can be detectedand cell colonies containing the functional transcription factor can beisolated and used to obtain the cloned gene which encodes the proteinwhich interacts with the transporter protein.

[0109] This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model. For example, an agent identified asdescribed herein (e.g., a transporter-modulating agent, an antisensetransporter nucleic acid molecule, a transporter-specific antibody, or atransporterbinding partner) can be used in an animal or other model todetermine the efficacy, toxicity, or side effects of treatment with suchan agent. Alternatively, an agent identified as described herein can beused in an animal or other model to determine the mechanism of action ofsuch an agent. Furthermore, this invention pertains to uses of novelagents identified by the above-described screening assays for treatmentsas described herein.

[0110] The transporter proteins of the present invention are also usefulto provide a target for diagnosing a disease or predisposition todisease mediated by the peptide. Accordingly, the invention providesmethods for detecting the presence, or levels of, the protein (orencoding mRNA) in a cell, tissue, or organism. Experimental data asprovided in FIG. 1 indicates expression in humans in the kidney andlung, as well as in kidney tumors. The method involves contacting abiological sample with a compound capable of interacting with thetransporter protein such that the interaction can be detected. Such anassay can be provided in a single detection format or a multi-detectionformat such as an antibody chip array.

[0111] One agent for detecting a protein in a sample is an antibodycapable of selectively binding to protein. A biological sample includestissues, cells and biological fluids isolated from a subject, as well astissues, cells and fluids present within a subject.

[0112] The peptides of the present invention also provide targets fordiagnosing active protein activity, disease, or predisposition todisease, in a patient having a variant peptide, particularly activitiesand conditions that are known for other members of the family ofproteins to which the present one belongs. Thus, the peptide can beisolated from a biological sample and assayed for the presence of agenetic mutation that results in aberrant peptide. This includes aminoacid substitution, deletion, insertion, rearrangement, (as the result ofaberrant splicing events), and inappropriate post-translationalmodification. Analytic methods include altered electrophoretic mobility,altered tryptic peptide digest, altered transporter activity incell-based or cell-free assay, alteration in ligand or antibody-bindingpattern, altered isoelectric point, direct amino acid sequencing, andany other of the known assay techniques useful for detecting mutationsin a protein. Such an assay can be provided in a single detection formator a multi-detection format such as an antibody chip array.

[0113] In vitro techniques for detection of peptide include enzymelinked immunosorbent assays (ELISAs), Western blots,immunoprecipitations and immunofluorescence using a detection reagent,such as an antibody or protein binding agent. Alternatively, the peptidecan be detected in vivo in a subject by introducing into the subject alabeled anti-peptide antibody or other types of detection agent. Forexample, the antibody can be labeled with a radioactive marker whosepresence and location in a subject can be detected by standard imagingtechniques. Particularly useful are methods that detect the allelicvariant of a peptide expressed in a subject and methods which detectfragments of a peptide in a sample.

[0114] The peptides are also useful in pharmacogenomic analysis.Pharmacogenomics deal with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, e.g., Eichelbaum, M. (Clin. Exp.Pharmacol. Physiol. 23(10-11):983-985 (1996)), and Linder, M. W. (Clin.Chem. 43(2):254-266 (1997)). The clinical outcomes of these variationsresult in severe toxicity of therapeutic drugs in certain individuals ortherapeutic failure of drugs in certain individuals as a result ofindividual variation in metabolism. Thus, the genotype of the individualcan determine the way a therapeutic compound acts on the body or the waythe body metabolizes the compound. Further, the activity of drugmetabolizing enzymes effects both the intensity and duration of drugaction. Thus, the pharmacogenomics of the individual permit theselection of effective compounds and effective dosages of such compoundsfor prophylactic or therapeutic treatment based on the individual'sgenotype. The discovery of genetic polymorphisms in some drugmetabolizing enzymes has explained why some patients do not obtain theexpected drug effects, show an exaggerated drug effect, or experienceserious toxicity from standard drug dosages. Polymorphisms can beexpressed in the phenotype of the extensive metabolizer and thephenotype of the poor metabolizer. Accordingly, genetic polymorphism maylead to allelic protein variants of the transporter protein in which oneor more of the transporter functions in one population is different fromthose in another population. The peptides thus allow a target toascertain a genetic predisposition that can affect treatment modality.Thus, in a ligand-based treatment, polymorphism may give rise to aminoterminal extracellular domains and/or other ligand-binding regions thatare more or less active in ligand binding, and transporter activation.Accordingly, ligand dosage would necessarily be modified to maximize thetherapeutic effect within a given population containing a polymorphism.As an alternative to genotyping, specific polymorphic peptides could beidentified.

[0115] The peptides are also useful for treating a disordercharacterized by an absence of, inappropriate, or unwanted expression ofthe protein. Experimental data as provided in FIG. 1 indicatesexpression in humans in the kidney and lung, as well as in kidneytumors. Accordingly, methods for treatment include the use of thetransporter protein or fragments.

[0116] Antibodies

[0117] The invention also provides antibodies that selectively bind toone of the peptides of the present invention, a protein comprising sucha peptide, as well as variants and fragments thereof. As used herein, anantibody selectively binds a target peptide when it binds the targetpeptide and does not significantly bind to unrelated proteins. Anantibody is still considered to selectively bind a peptide even if italso binds to other proteins that are not substantially homologous withthe target peptide so long as such proteins share homology with afragment or domain of the peptide target of the antibody. In this case,it would be understood that antibody binding to the peptide is stillselective despite some degree of cross-reactivity.

[0118] As used herein, an antibody is defined in terms consistent withthat recognized within the art: they are multi-subunit proteins producedby a mammalian organism in response to an antigen challenge. Theantibodies of the present invention include polyclonal antibodies andmonoclonal antibodies, as well as fragments of such antibodies,including, but not limited to, Fab or F(ab′)₂, and Fv fragments.

[0119] Many methods are known for generating and/or identifyingantibodies to a given target peptide. Several such methods are describedby Harlow, Antibodies, Cold Spring Harbor Press, (1989).

[0120] In general, to generate antibodies, an isolated peptide is usedas an immunogen and is administered to a mammalian organism, such as arat, rabbit or mouse. The full-length protein, an antigenic peptidefragment or a fusion protein can be used. Particularly importantfragments are those covering functional domains, such as the domainsidentified in FIG. 2, and domain of sequence homology or divergenceamongst the family, such as those that can readily be identified usingprotein alignment methods and as presented in the Figures.

[0121] Antibodies are preferably prepared from regions or discretefragments of the transporter proteins. Antibodies can be prepared fromany region of the peptide as described herein. However, preferredregions will include those involved in function/activity and/ortransporter/binding partner interaction. FIG. 2 can be used to identifyparticularly important regions while sequence alignment can be used toidentify conserved and unique sequence fragments.

[0122] An antigenic fragment will typically comprise at least 8contiguous amino acid residues. The antigenic peptide can comprise,however, at least 10, 12, 14, 16 or more amino acid residues. Suchfragments can be selected on a physical property, such as fragmentscorrespond to regions that are located on the surface of the protein,e.g., hydrophilic regions or can be selected based on sequenceuniqueness (see FIG. 2).

[0123] Detection on an antibody of the present invention can befacilitated by coupling (i.e., physically linking) the antibody to adetectable substance. Examples of detectable substances include variousenzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, and radioactive materials. Examplesof suitable enzymes include horseradish peroxidase, alkalinephosphatase, β-galactosidase, or acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or³H.

[0124] Antibody Uses

[0125] The antibodies can be used to isolate one of the proteins of thepresent invention by standard techniques, such as affinitychromatography or immunoprecipitation. The antibodies can facilitate thepurification of the natural protein from cells and recombinantlyproduced protein expressed in host cells. In addition, such antibodiesare useful to detect the presence of one of the proteins of the presentinvention in cells or tissues to determine the pattern of expression ofthe protein among various tissues in an organism and over the course ofnormal development. Experimental data as provided in FIG. 1 indicatesthat the transporter proteins of the present invention are expressed inhumans in both normal kidney tissue and kidney tumors, as indicated byvirtual northern blot analysis. In addition, PCR-based tissue screeningpanels indicate expression in the human lung. Further, such antibodiescan be used to detect protein in situ, in vitro, or in a cell lysate orsupernatant in order to evaluate the abundance and pattern ofexpression. Also, such antibodies can be used to assess abnormal tissuedistribution or abnormal expression during development or progression ofa biological condition. Antibody detection of circulating fragments ofthe full length protein can be used to identify turnover.

[0126] Further, the antibodies can be used to assess expression indisease states such as in active stages of the disease or in anindividual with a predisposition toward disease related to the protein'sfunction. When a disorder is caused by an inappropriate tissuedistribution, developmental expression, level of expression of theprotein, or expressed/processed form, the antibody can be preparedagainst the normal protein. Experimental data as provided in FIG. 1indicates expression in humans in the kidney and lung, as well as inkidney tumors. If a disorder is characterized by a specific mutation inthe protein, antibodies specific for this mutant protein can be used toassay for the presence of the specific mutant protein.

[0127] The antibodies can also be used to assess normal and aberrantsubcellular localization of cells in the various tissues in an organism.Experimental data as provided in FIG. 1 indicates expression in humansin the kidney and lung, as well as in kidney tumors. The diagnostic usescan be applied, not only in genetic testing, but also in monitoring atreatment modality. Accordingly, where treatment is ultimately aimed atcorrecting expression level or the presence of aberrant sequence andaberrant tissue distribution or developmental expression, antibodiesdirected against the protein or relevant fragments can be used tomonitor therapeutic efficacy.

[0128] Additionally, antibodies are useful in pharmacogenornic analysis.Thus, antibodies prepared against polymorphic proteins can be used toidentify individuals that require modified treatment modalities. Theantibodies are also useful as diagnostic tools as an immunologicalmarker for aberrant protein analyzed by electrophoretic mobility,isoelectric point, tryptic peptide digest, and other physical assaysknown to those in the art.

[0129] The antibodies are also useful for tissue typing. Experimentaldata as provided in FIG. 1 indicates expression in humans in the kidneyand lung, as well as in kidney tumors. Thus, where a specific proteinhas been correlated with expression in a specific tissue, antibodiesthat are specific for this protein can be used to identify a tissuetype.

[0130] The antibodies are also useful for inhibiting protein function,for example, blocking the binding of the transporter peptide to abinding partner such as a ligand or protein binding partner. These usescan also be applied in a therapeutic context in which treatment involvesinhibiting the protein's function. An antibody can be used, for example,to block binding, thus modulating (agonizing or antagonizing) thepeptides activity. Antibodies can be prepared against specific fragmentscontaining sites required for function or against intact protein that isassociated with a cell or cell membrane. See FIG. 2 for structuralinformation relating to the proteins of the present invention.

[0131] The invention also encompasses kits for using antibodies todetect the presence of a protein in a biological sample. The kit cancomprise antibodies such as a labeled or labelable antibody and acompound or agent for detecting protein in a biological sample; meansfor determining the amount of protein in the sample; means for comparingthe amount of protein in the sample with a standard; and instructionsfor use. Such a kit can be supplied to detect a single protein orepitope or can be configured to detect one of a multitude of epitopes,such as in an antibody detection array. Arrays are described in detailbelow for nucleic acid arrays and similar methods have been developedfor antibody arrays.

[0132] Nucleic Acid Molecules

[0133] The present invention further provides isolated nucleic acidmolecules that encode a transporter peptide or protein of the presentinvention (cDNA, transcript and genomic sequence). Such nucleic acidmolecules will consist of, consist essentially of, or comprise anucleotide sequence that encodes one of the transporter peptides of thepresent invention, an allelic variant thereof, or an ortholog or paralogthereof.

[0134] As used herein, an “isolated” nucleic acid molecule is one thatis separated from other nucleic acid present in the natural source ofthe nucleic acid. Preferably, an “isolated” nucleic acid is free ofsequences that naturally flank the nucleic acid (i.e., sequences locatedat the 5′ and 3′ ends of the nucleic acid) in the genomic DNA of theorganism from which the nucleic acid is derived. However, there can besome flanking nucleotide sequences, for example up to about 5 KB, 4 KB,3 KB, 2 KB, or 1 KB or less, particularly contiguous peptide encodingsequences and peptide encoding sequences within the same gene butseparated by introns in the genomic sequence. The important point isthat the nucleic acid is isolated from remote and unimportant flankingsequences such that it can be subjected to the specific manipulationsdescribed herein such as recombinant expression, preparation of probesand primers, and other uses specific to the nucleic acid sequences.

[0135] Moreover, an “isolated” nucleic acid molecule, such as atranscript/cDNA molecule, can be substantially free of other cellularmaterial, or culture medium when produced by recombinant techniques, orchemical precursors or other chemicals when chemically synthesized.However, the nucleic acid molecule can be fused to other coding orregulatory sequences and still be considered isolated.

[0136] For example, recombinant DNA molecules contained in a vector areconsidered isolated. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified (partially or substantially) DNA molecules in solution.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe isolated DNA molecules of the present invention. Isolated nucleicacid molecules according to the present invention further include suchmolecules produced synthetically.

[0137] Accordingly, the present invention provides nucleic acidmolecules that consist of the nucleotide sequence shown in FIG. 1 or 3(SEQ ID NO: 1, transcript sequence and SEQ ID NO: 3, genomic sequence),or any nucleic acid molecule that encodes the protein provided in FIG.2, SEQ ID NO: 2. A nucleic acid molecule consists of a nucleotidesequence when the nucleotide sequence is the complete nucleotidesequence of the nucleic acid molecule.

[0138] The present invention further provides nucleic acid moleculesthat consist essentially of the nucleotide sequence shown in FIG. 1 or 3(SEQ ID NO: 1, transcript sequence and SEQ ID NO: 3, genomic sequence),or any nucleic acid molecule that encodes the protein provided in FIG.2, SEQ ID NO: 2. A nucleic acid molecule consists essentially of anucleotide sequence when such a nucleotide sequence is present with onlya few additional nucleic acid residues in the final nucleic acidmolecule.

[0139] The present invention further provides nucleic acid moleculesthat comprise the nucleotide sequences shown in FIG. 1 or 3 (SEQ ID NO:1, transcript sequence and SEQ ID NO: 3, genomic sequence), or anynucleic acid molecule that encodes the protein provided in FIG. 2, SEQID NO: 2. A nucleic acid molecule comprises a nucleotide sequence whenthe nucleotide sequence is at least part of the final nucleotidesequence of the nucleic acid molecule. In such a fashion, the nucleicacid molecule can be only the nucleotide sequence or have additionalnucleic acid residues, such as nucleic acid residues that are naturallyassociated with it or heterologous nucleotide sequences. Such a nucleicacid molecule can have a few additional nucleotides or can compriseseveral hundred or more additional nucleotides. A brief description ofhow various types of these nucleic acid molecules can be readilymade/isolated is provided below.

[0140] In FIGS. 1 and 3, both coding and non-coding sequences areprovided. Because of the source of the present invention, humans genomicsequence (FIG. 3) and cDNA/transcript sequences (FIG. 1), the nucleicacid molecules in the Figures will contain genomic intronic sequences,5′ and 3′ non-coding sequences, gene regulatory regions and non-codingintergenic sequences. In general such sequence features are either notedin FIGS. 1 and 3 or can readily be identified using computational toolsknown in the art. As discussed below, some of the non-coding regions,particularly gene regulatory elements such as promoters, are useful fora variety of purposes, e.g. control of heterologous gene expression,target for identifying gene activity modulating compounds, and areparticularly claimed as fragments of the genomic sequence providedherein.

[0141] The isolated nucleic acid molecules can encode the mature proteinplus additional amino or carboxyl-terminal amino acids, or amino acidsinterior to the mature peptide (when the mature form has more than onepeptide chain, for instance). Such sequences may play a role inprocessing of a protein from precursor to a mature form, facilitateprotein trafficking, prolong or shorten protein half-life or facilitatemanipulation of a protein for assay or production, anong other things.As generally is the case in situ, the additional amino acids may beprocessed away from the mature protein by cellular enzymes.

[0142] As mentioned above, the isolated nucleic acid molecules include,but are not limited to, the sequence encoding the transporter peptidealone, the sequence encoding the mature peptide and additional codingsequences, such as a leader or secretory sequence (e.g., a pre-pro orpro-protein sequence), the sequence encoding the mature peptide, with orwithout the additional coding sequences, plus additional non-codingsequences, for example introns and non-coding 5′ and 3′ sequences suchas transcribed but non-translated sequences that play a role intranscription, mRNA processing (including splicing and polyadenylationsignals), ribosome binding and stability of mRNA. In addition, thenucleic acid molecule may be fused to a marker sequence encoding, forexample, a peptide that facilitates purification.

[0143] Isolated nucleic acid molecules can be in the form of RNA, suchas mRNA, or in the form DNA, including cDNA and genomic DNA obtained bycloning or produced by chemical synthetic techniques or by a combinationthereof. The nucleic acid, especially DNA, can be double-stranded orsingle-stranded. Single-stranded nucleic acid can be the coding strand(sense strand) or the non-coding strand (anti-sense strand).

[0144] The invention further provides nucleic acid molecules that encodefragments of the peptides of the present invention as well as nucleicacid molecules that encode obvious variants of the transporter proteinsof the present invention that are described above. Such nucleic acidmolecules may be naturally occurring, such as allelic variants (samelocus), paralogs (different locus), and orthologs (different organism),or may be constructed by recombinant DNA methods or by chemicalsynthesis. Such non-naturally occurring variants may be made bymutagenesis techniques, including those applied to nucleic acidmolecules, cells, or organisms. Accordingly, as discussed above, thevariants can contain nucleotide substitutions, deletions, inversions andinsertions. Variation can occur in either or both the coding andnon-coding regions. The variations can produce both conservative andnon-conservative amino acid substitutions.

[0145] The present invention further provides non-coding fragments ofthe nucleic acid molecules provided in FIGS. 1 and 3. Preferrednon-coding fragments include, but are not limited to, promotersequences, enhancer sequences, gene modulating sequences and genetermination sequences. Such fragments are useful in controllingheterologous gene expression and in developing screens to identifygene-modulating agents. A promoter can readily be identified as being 5′to the ATG start site in the genomic sequence provided in FIG. 3.

[0146] A fragment comprises a contiguous nucleotide sequence greaterthan 12 or more nucleotides. Further, a fragment could at least 30, 40,50, 100, 250 or 500 nucleotides in length. The length of the fragmentwill be based on its intended use. For example, the fragment can encodeepitope bearing regions of the peptide, or can be useful as DNA probesand primers. Such fragments can be isolated using the known nucleotidesequence to synthesize an oligonucleotide probe. A labeled probe canthen be used to screen a cDNA library, genomic DNA library, or mRNA toisolate nucleic acid corresponding to the coding region. Further,primers can be used in PCR reactions to clone specific regions of gene.

[0147] A probe/primer typically comprises substantially a purifiedoligonucleotide or oligonucleotide pair. The oligonucleotide typicallycomprises a region of nucleotide sequence that hybridizes understringent conditions to at least about 12, 20, 25, 40, 50 or moreconsecutive nucleotides.

[0148] Orthologs, homologs, and allelic variants can be identified usingmethods well known in the art. As described in the Peptide Section,these variants comprise a nucleotide sequence encoding a peptide that istypically 60-70%, 70-80%, 80-90%, and more typically at least about90-95% or more homologous to the nucleotide sequence shown in the Figuresheets or a fragment of this sequence. Such nucleic acid molecules canreadily be identified as being able to hybridize under moderate tostringent conditions, to the nucleotide sequence shown in the Figuresheets or a fragment of the sequence. Allelic variants can readily bedetermined by genetic locus of the encoding gene. The gene encoding thenovel transporter protein of the present invention is located on agenome component that has been mapped to human chromosome 8 (asindicated in FIG. 3), which is supported by multiple lines of evidence,such as STS and BAC map data.

[0149]FIG. 3 provides information on SNPs that have been found in thegene encoding the transporter protein of the present invention. SNPswere identified at 77 different nucleotide positions. These SNPs,particularly the SNPs located in the first intron, may affectcontrol/regulatory elements.

[0150] As used herein, the term “hybridizes under stringent conditions”is intended to describe conditions for hybridization and washing underwhich nucleotide sequences encoding a peptide at least 60-70% homologousto each other typically remain hybridized to each other. The conditionscan be such that sequences at least about 60%, at least about 70%, or atleast about 80% or more homologous to each other typically remainhybridized to each other. Such stringent conditions are known to thoseskilled in the art and can be found in Current Protocols in MolecularBiology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. One example ofstringent hybridization conditions are hybridization in 6X sodiumchloride/sodium citrate (SSC) at about 45C, followed by one or morewashes in 0.2 X SSC, 0.1% SDS at 50-65C. Examples of moderate to lowstringency hybridization conditions are well known in the art.

[0151] Nucleic Acid Molecule Uses

[0152] The nucleic acid molecules of the present invention are usefulfor probes, primers, chemical intermediates, and in biological assays.The nucleic acid molecules are useful as a hybridization probe formessenger RNA, transcript/cDNA and genomic DNA to isolate full-lengthcDNA and genomic clones encoding the peptide described in FIG. 2 and toisolate cDNA and genomic clones that correspond to variants (alleles,orthologs, etc.) producing the same or related peptides shown in FIG. 2.As illustrated in FIG. 3, SNPs were identified at 77 differentnucleotide positions.

[0153] The probe can correspond to any sequence along the entire lengthof the nucleic acid molecules provided in the Figures. Accordingly, itcould be derived from 5′ noncoding regions, the coding region, and 3′noncoding regions. However, as discussed, fragments are not to beconstrued as encompassing fragments disclosed prior to the presentinvention.

[0154] The nucleic acid molecules are also useful as primers for PCR toamplify any given region of a nucleic acid molecule and are useful tosynthesize antisense molecules of desired length and sequence.

[0155] The nucleic acid molecules are also useful for constructingrecombinant vectors. Such vectors include expression vectors thatexpress a portion of, or all of, the peptide sequences. Vectors alsoinclude insertion vectors, used to integrate into another nucleic acidmolecule sequence, such as into the cellular genome, to alter in situexpression of a gene and/or gene product. For example, an endogenouscoding sequence can be replaced via homologous recombination with all orpart of the coding region containing one or more specifically introducedmutations.

[0156] The nucleic acid molecules are also useful for expressingantigenic portions of the proteins.

[0157] The nucleic acid molecules are also useful as probes fordetermining the chromosomal positions of the nucleic acid molecules bymeans of in situ hybridization methods. The gene encoding the noveltransporter protein of the present invention is located on a genomecomponent that has been mapped to human chromosome 8 (as indicated inFIG. 3), which is supported by multiple lines of evidence, such as STSand BAC map data.

[0158] The nucleic acid molecules are also useful in making vectorscontaining the gene regulatory regions of the nucleic acid molecules ofthe present invention.

[0159] The nucleic acid molecules are also useful for designingribozymes corresponding to all, or a part, of the mRNA produced from thenucleic acid molecules described herein.

[0160] The nucleic acid molecules are also useful for making vectorsthat express part, or all, of the peptides.

[0161] The nucleic acid molecules are also useful for constructing hostcells expressing a part, or all, of the nucleic acid molecules andpeptides.

[0162] The nucleic acid molecules are also useful for constructingtransgenic animals expressing all, or a part, of the nucleic acidmolecules and peptides.

[0163] The nucleic acid molecules are also useful as hybridizationprobes for determining the presence, level, form and distribution ofnucleic acid expression. Experimental data as provided in FIG. 1indicates that the transporter proteins of the present invention areexpressed in humans in both normal kidney tissue and kidney tumors, asindicated by virtual northern blot analysis. In addition, PCR-basedtissue screening panels indicate expression in the human lung.

[0164] Accordingly, the probes can be used to detect the presence of, orto determine levels of, a specific nucleic acid molecule in cells,tissues, and in organisms. The nucleic acid whose level is determinedcan be DNA or RNA. Accordingly, probes corresponding to the peptidesdescribed herein can be used to assess expression and/or gene copynumber in a given cell, tissue, or organism. These uses are relevant fordiagnosis of disorders involving an increase or decrease in transporterprotein expression relative to normal results.

[0165] In vitro techniques for detection of mRNA include Northernhybridizations and in situ hybridizations. In vitro techniques fordetecting DNA include Southern hybridizations and in situ hybridization.

[0166] Probes can be used as a part of a diagnostic test kit foridentifying cells or tissues that express a transporter protein, such asby measuring a level of a transporter-encoding nucleic acid in a sampleof cells from a subject e.g., mRNA or genomic DNA, or determining if atransporter gene has been mutated. Experimental data as provided in FIG.1 indicates that the transporter proteins of the present invention areexpressed in humans in both normal kidney tissue and kidney tumors, asindicated by virtual northern blot analysis. In addition, PCR-basedtissue screening panels indicate expression in the human lung.

[0167] Nucleic acid expression assays are useful for drug screening toidentify compounds that modulate transporter nucleic acid expression.

[0168] The invention thus provides a method for identifying a compoundthat can be used to treat a disorder associated with nucleic acidexpression of the transporter gene, particularly biological andpathological processes that are mediated by the transporter in cells andtissues that express it. Experimental data as provided in FIG. 1indicates expression in humans in the kidney and lung, as well as inkidney tumors. The method typically includes assaying the ability of thecompound to modulate the expression of the transporter nucleic acid andthus identifying a compound that can be used to treat a disordercharacterized by undesired transporter nucleic acid expression. Theassays can be performed in cell-based and cell-free systems. Cell-basedassays include cells naturally expressing the transporter nucleic acidor recombinant cells genetically engineered to express specific nucleicacid sequences.

[0169] The assay for transporter nucleic acid expression can involvedirect assay of nucleic acid levels, such as mRNA levels, or oncollateral compounds involved in the signal pathway. Further, theexpression of genes that are up- or down-regulated in response to thetransporter protein signal pathway can also be assayed. In thisembodiment the regulatory regions of these genes can be operably linkedto a reporter gene such as luciferase.

[0170] Thus, modulators of transporter gene expression can be identifiedin a method wherein a cell is contacted with a candidate compound andthe expression of mRNA determined. The level of expression oftransporter mRNA in the presence of the candidate compound is comparedto the level of expression of transporter mRNA in the absence of thecandidate compound. The candidate compound can then be identified as amodulator of nucleic acid expression based on this comparison and beused, for example to treat a disorder characterized by aberrant nucleicacid expression. When expression of mRNA is statistically significantlygreater in the presence of the candidate compound than in its absence,the candidate compound is identified as a stimulator of nucleic acidexpression. When nucleic acid expression is statistically significantlyless in the presence of the candidate compound than in its absence, thecandidate compound is identified as an inhibitor of nucleic acidexpression.

[0171] The invention further provides methods of treatment, with thenucleic acid as a target, using a compound identified through drugscreening as a gene modulator to modulate transporter nucleic acidexpression in cells and tissues that express the transporter.Experimental data as provided in FIG. 1 indicates that the transporterproteins of the present invention are expressed in humans in both normalkidney tissue and kidney tumors, as indicated by virtual northern blotanalysis. In addition, PCR-based tissue screening panels indicateexpression in the human lung. Modulation includes both up-regulation(i.e. activation or agonization) or down-regulation (suppression orantagonization) or nucleic acid expression.

[0172] Alternatively, a modulator for transporter nucleic acidexpression can be a small molecule or drug identified using thescreening assays described herein as long as the drug or small moleculeinhibits the transporter nucleic acid expression in the cells andtissues that express the protein. Experimental data as provided in FIG.1 indicates expression in humans in the kidney and lung, as well as inkidney tumors.

[0173] The nucleic acid molecules are also useful for monitoring theeffectiveness of modulating compounds on the expression or activity ofthe transporter gene in clinical trials or in a treatment regimen. Thus,the gene expression pattern can serve as a barometer for the continuingeffectiveness of treatment with the compound, particularly withcompounds to which a patient can develop resistance. The gene expressionpattern can also serve as a marker indicative of a physiologicalresponse of the affected cells to the compound. Accordingly, suchmonitoring would allow either increased administration of the compoundor the administration of alternative compounds to which the patient hasnot become resistant. Similarly, if the level of nucleic acid expressionfalls below a desirable level, administration of the compound could beconmmensurately decreased.

[0174] The nucleic acid molecules are also useful in diagnostic assaysfor qualitative changes in transporter nucleic acid expression, andparticularly in qualitative changes that lead to pathology. The nucleicacid molecules can be used to detect mutations in transporter genes andgene expression products such as mRNA. The nucleic acid molecules can beused as hybridization probes to detect naturally occurring geneticmutations in the transporter gene and thereby to determine whether asubject with the mutation is at risk for a disorder caused by themutation. Mutations include deletion, addition, or substitution of oneor more nucleotides in the gene, chromosomal rearrangement, such asinversion or transposition, modification of genomic DNA, such asaberrant methylation patterns or changes in gene copy number, such asamplification. Detection of a mutated form of the transporter geneassociated with a dysfunction provides a diagnostic tool for an activedisease or susceptibility to disease when the disease results fromoverexpression, underexpression, or altered expression of a transporterprotein.

[0175] Individuals carrying mutations in the transporter gene can bedetected at the nucleic acid level by a variety of techniques. FIG. 3provides information on SNPs that have been found in the gene encodingthe transporter protein of the present invention. SNPs were identifiedat 77 different nucleotide positions. These SNPs, particularly the SNPslocated in the first intron, may affect control/regulatory elements. Thegene encoding the novel transporter protein of the present invention islocated on a genome component that has been mapped to human chromosome 8(as indicated in FIG. 3), which is supported by multiple lines ofevidence, such as STS and BAC map data. Genomic DNA can be analyzeddirectly or can be amplified by using PCR prior to analysis. RNA or cDNAcan be used in the same way. In some uses, detection of the mutationinvolves the use of a probe/primer in a polymerase chain reaction (PCR)(see, e.g. U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCRor RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see,e.g., Landegran et al., Science 241:1077-1080 (1988); and Nakazawa etal., PNAS 91:360-364 (1994)), the latter of which can be particularlyuseful for detecting point mutations in the gene (see Abravaya et al.,Nucleic Acids Res. 23:675-682 (1995)). This method can include the stepsof collecting a sample of cells from a patient, isolating nucleic acid(e.g., genomic, mRNA or both) from the cells of the sample, contactingthe nucleic acid sample with one or more primers which specificallyhybridize to a gene under conditions such that hybridization andamplification of the gene (if present) occurs, and detecting thepresence or absence of an amplification product, or detecting the sizeof the amplification product and comparing the length to a controlsample. Deletions and insertions can be detected by a change in size ofthe amplified product compared to the normal genotype. Point mutationscan be identified by hybridizing amplified DNA to normal RNA orantisense DNA sequences.

[0176] Alternatively, mutations in a transporter gene can be directlyidentified, for example, by alterations in restriction enzyme digestionpatterns determined by gel electrophoresis.

[0177] Further, sequence-specific ribozymes (U.S. Pat. No. 5,498,531)can be used to score for the presence of specific mutations bydevelopment or loss of a ribozyme cleavage site. Perfectly matchedsequences can be distinguished from mismatched sequences by nucleasecleavage digestion assays or by differences in melting temperature.

[0178] Sequence changes at specific locations can also be assessed bynuclease protection assays such as RNase and SI protection or thechemical cleavage method. Furthermore, sequence differences between amutant transporter gene and a wild-type gene can be determined by directDNA sequencing. A variety of automated sequencing procedures can beutilized when performing the diagnostic assays (Naeve, C. W., (1995)Biotechniques 19:448), including sequencing by mass spectrometry (see,e.g., PCT International Publication No. WO 94/16101; Cohen et al., Adv.Chromatogr. 36:127-162 (1996); and Griffin et al., Appl. Biochem.Biotechnol. 38:147-159 (1993)).

[0179] Other methods for detecting mutations in the gene include methodsin which protection from cleavage agents is used to detect mismatchedbases in RNA/RNA or RNA/DNA duplexes (Myers et al., Science 230:1242(1985)); Cotton et al., PNAS85:4397 (1988); Saleeba et al., Meth.Enzymol. 217:286-295 (1992)), electrophoretic mobility of mutant andwild type nucleic acid is compared (Orita et al., PNAS 86:2766 (1989);Cotton et al., Mutat. Res. 285:125-144 (1993); and Hayashi et al.,Genet. Anal. Tech. Appl. 9:73-79 (1992)), and movement of mutant orwild-type fragments in polyacrylamide gels containing a gradient ofdenaturant is assayed using denaturing gradient gel electrophoresis(Myers et al., Nature 313:495 (1985)). Examples of other techniques fordetecting point mutations include selective oligonucleotidehybridization, selective amplification, and selective primer extension.

[0180] The nucleic acid molecules are also useful for testing anindividual for a genotype that while not necessarily causing thedisease, nevertheless affects the treatment modality. Thus, the nucleicacid molecules can be used to study the relationship between anindividual's genotype and the individual's response to a compound usedfor treatment (pharmacogenomic relationship). Accordingly, the nucleicacid molecules described herein can be used to assess the mutationcontent of the transporter gene in an individual in order to select anappropriate compound or dosage regimen for treatment. FIG. 3 providesinformation on SNPs that have been found in the gene encoding thetransporter protein of the present invention. SNPs were identified at 77different nucleotide positions. These SNPs, particularly the SNPslocated in the first intron, may affect control/regulatory elements.

[0181] Thus nucleic acid molecules displaying genetic variations thataffect treatment provide a diagnostic target that can be used to tailortreatment in an individual. Accordingly, the production of recombinantcells and animals containing these polymorphisms allow effectiveclinical design of treatment compounds and dosage regimens.

[0182] The nucleic acid molecules are thus useful as antisenseconstructs to control transporter gene expression in cells, tissues, andorganisms. A DNA antisense nucleic acid molecule is designed to becomplementary to a region of the gene involved in transcription,preventing transcription and hence production of transporter protein. Anantisense RNA or DNA nucleic acid molecule would hybridize to the mRNAand thus block translation of mRNA into transporter protein.

[0183] Alternatively, a class of antisense molecules can be used toinactivate mRNA in order to decrease expression of transporter nucleicacid. Accordingly, these molecules can treat a disorder characterized byabnormal or undesired transporter nucleic acid expression. Thistechnique involves cleavage by means of ribozymes containing nucleotidesequences complementary to one or more regions in the mRNA thatattenuate the ability of the mRNA to be translated. Possible regionsinclude coding regions and particularly coding regions corresponding tothe catalytic and other functional activities of the transporterprotein, such as ligand binding.

[0184] The nucleic acid molecules also provide vectors for gene therapyin patients containing cells that are aberrant in transporter geneexpression. Thus, recombinant cells, which include the patient's cellsthat have been engineered ex vivo and returned to the patient, areintroduced into an individual where the cells produce the desiredtransporter protein to treat the individual.

[0185] The invention also encompasses kits for detecting the presence ofa transporter nucleic acid in a biological sample. Experimental data asprovided in FIG. 1 indicates that the transporter proteins of thepresent invention are expressed in humans in both normal kidney tissueand kidney tumors, as indicated by virtual northern blot analysis. Inaddition, PCR-based tissue screening panels indicate expression in thehuman lung. For example, the kit can comprise reagents such as a labeledor labelable nucleic acid or agent capable of detecting transporternucleic acid in a biological sample; means for determining the amount oftransporter nucleic acid in the sample; and means for comparing theamount of transporter nucleic acid in the sample with a standard. Thecompound or agent can be packaged in a suitable container. The kit canfurther comprise instructions for using the kit to detect transporterprotein mRNA or DNA.

[0186] Nucleic Acid Arrays

[0187] The present invention further provides nucleic acid detectionkits, such as arrays or microarrays of nucleic acid molecules that arebased on the sequence information provided in FIGS. 1 and 3 (SEQ ID NOS:1 and 3).

[0188] As used herein “Arrays” or “Microarrays” refers to an array ofdistinct polynucleotides or oligonucleotides synthesized on a substrate,such as paper, nylon or other type of membrane, filter, chip, glassslide, or any other suitable solid support. In one embodiment, themicroarray is prepared and used according to the methods described inU.S. Pat. No. 5,837,832, Chee et al., PCT application WO95/11995 (Cheeet al.), Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) andSchena, M. et al. (1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all ofwhich are incorporated herein in their entirety by reference. In otherembodiments, such arrays are produced by the methods described by Brownet al., U.S. Pat. No. 5,807,522.

[0189] The microarray or detection kit is preferably composed of a largenumber of unique, single-stranded nucleic acid sequences, usually eithersynthetic antisense oligonucleotides or fragments of cDNAs, fixed to asolid support. The oligonucleotides are preferably about 6-60nucleotides in length, more preferably 15-30 nucleotides in length, andmost preferably about 20-25 nucleotides in length. For a certain type ofmicroarray or detection kit, it may be preferable to useoligonucleotides that are only 7-20 nucleotides in length. Themicroarray or detection kit may contain oligonucleotides that cover theknown 5′, or 3′, sequence, sequential oligonucleotides that cover thefull length sequence; or unique oligonucleotides selected fromparticular areas along the length of the sequence. Polynucleotides usedin the microarray or detection kit may be oligonucleotides that arespecific to a gene or genes of interest.

[0190] In order to produce oligonucleotides to a known sequence for amicroarray or detection kit, the gene(s) of interest (or an ORFidentified from the contigs of the present invention) is typicallyexamined using a computer algorithm which starts at the 5′ or at the 3′end of the nucleotide sequence. Typical algorithms will then identifyoligomers of defined length that are unique to the gene, have a GCcontent within a range suitable for hybridization, and lack predictedsecondary structure that may interfere with hybridization. In certainsituations it may be appropriate to use pairs of oligonucleotides on amicroarray or detection kit. The “pairs” will be identical, except forone nucleotide that preferably is located in the center of the sequence.The second oligonucleotide in the pair (mismatched by one) serves as acontrol. The number of oligonucleotide pairs may range from two to onemillion. The oligomers are synthesized at designated areas on asubstrate using a light-directed chemical process. The substrate may bepaper, nylon or other type of membrane, filter, chip, glass slide or anyother suitable solid support.

[0191] In another aspect, an oligonucleotide may be synthesized on thesurface of the substrate by using a chemical coupling procedure and anink jet application apparatus, as described in PCT applicationWO95/251116 (Baldeschweiler et al.) which is incorporated herein in itsentirety by reference. In another aspect, a “gridded” array analogous toa dot (or slot) blot may be used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array, suchas those described above, may be produced by hand or by using availabledevices (slot blot or dot blot apparatus), materials (any suitable solidsupport), and machines (including robotic instruments), and may contain8, 24, 96, 384, 1536, 6144 or more oligonucleotides, or any other numberbetween two and one million which lends itself to the efficient use ofcommercially available instrumentation.

[0192] In order to conduct sample analysis using a microarray ordetection kit, the RNA or DNA from a biological sample is made intohybridization probes. The mRNA is isolated, and cDNA is produced andused as a template to make antisense RNA (aRNA). The aRNA is amplifiedin the presence of fluorescent nucleotides, and labeled probes areincubated with the microarray or detection kit so that the probesequences hybridize to complementary oligonucleotides of the microarrayor detection kit. Incubation conditions are adjusted so thathybridization occurs with precise complementary matches or with variousdegrees of less complementarity. After removal of nonhybridized probes,a scanner is used to determnine the levels and patterns of fluorescence.The scanned images are examined to determine degree of complementarityand the relative abundance of each oligonucleotide sequence on themicroarray or detection kit. The biological samples may be obtained fromany bodily fluids (such as blood, urine, saliva, phlegm, gastric juices,etc.), cultured cells, biopsies, or other tissue preparations. Adetection system may be used to measure the absence, presence, andamount of hybridization for all of the distinct sequencessimultaneously. This data may be used for large-scale correlationstudies on the sequences, expression patterns, mutations, variants, orpolymorphisms among samples.

[0193] Using such arrays, the present invention provides methods toidentify the expression of the transporter proteins/peptides of thepresent invention. In detail, such methods comprise incubating a testsample with one or more nucleic acid molecules and assaying for bindingof the nucleic acid molecule with components within the test sample.Such assays will typically involve arrays comprising many genes, atleast one of which is a gene of the present invention and or alleles ofthe transporter gene of the present invention. FIG. 3 providesinformation on SNPs that have been found in the gene encoding thetransporter protein of the present invention. SNPs were identified at 77different nucleotide positions. These SNPs, particularly the SNPslocated in the first intron, may affect control/regulatory elements.

[0194] Conditions for incubating a nucleic acid molecule with a testsample vary. Incubation conditions depend on the format employed in theassay, the detection methods employed, and the type and nature of thenucleic acid molecule used in the assay. One skilled in the art willrecognize that any one of the commonly available hybridization,amplification or array assay formats can readily be adapted to employthe novel fragments of the Human genome disclosed herein. Examples ofsuch assays can be found in Chard, T, An Introduction toRadioimmunoassay and Related Techniques, Elsevier Science Publishers,Amsterdam, The Netherlands (1986); Bullock, G. R. et al., Techniques inImmunocytochemistry, Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2(1983), Vol. 3 (1985); Tijssen, P., Practice and Theory of EnzymeImmunoassays: Laboratory Techniques in Biochemistry and MolecularBiology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985).

[0195] The test samples of the present invention include cells, proteinor membrane extracts of cells. The test sample used in theabove-described method will vary based on the assay format, nature ofthe detection method and the tissues, cells or extracts used as thesample to be assayed. Methods for preparing nucleic acid extracts or ofcells are well known in the art and can be readily be adapted in orderto obtain a sample that is compatible with the system utilized.

[0196] In another embodiment of the present invention, kits are providedwhich contain the necessary reagents to carry out the assays of thepresent invention.

[0197] Specifically, the invention provides a compartmentalized kit toreceive, in close confinement, one or more containers which comprises:(a) a first container comprising one of the nucleic acid molecules thatcan bind to a fragment of the Human genome disclosed herein; and (b) oneor more other containers comprising one or more of the following: washreagents, reagents capable of detecting presence of a bound nucleicacid.

[0198] In detail, a compartmentalized kit includes any kit in whichreagents are contained in separate containers. Such containers includesmall glass containers, plastic containers, strips of plastic, glass orpaper, or arraying material such as silica. Such containers allows oneto efficiently transfer reagents from one compartment to anothercompartment such that the samples and reagents are notcross-contaminated, and the agents or solutions of each container can beadded in a quantitative fashion from one compartment to another. Suchcontainers will include a container which will accept the test sample, acontainer which contains the nucleic acid probe, containers whichcontain wash reagents (such as phosphate buffered saline, Tris-buffers,etc.), and containers which contain the reagents used to detect thebound probe. One skilled in the art will readily recognize that thepreviously unidentified transporter gene of the present invention can beroutinely identified using the sequence information disclosed herein canbe readily incorporated into one of the established kit formats whichare well known in the art, particularly expression arrays.

[0199] Vectors/Host Cells

[0200] The invention also provides vectors containing the nucleic acidmolecules described herein. The tenn “vector” refers to a vehicle,preferably a nucleic acid molecule, which can transport the nucleic acidmolecules. When the vector is a nucleic acid molecule, the nucleic acidmolecules are covalently linked to the vector nucleic acid. With thisaspect of the invention, the vector includes a plasmid, single or doublestranded phage, a single or double stranded RNA or DNA viral vector, orartificial chromosome, such as a BAC, PAC, YAC, OR MAC.

[0201] A vector can be maintained in the host cell as anextrachromosomal element where itreplicates and produces additionalcopies of the nucleic acid molecules. Alternatively, the vector mayintegrate into the host cell genome and produce additional copies of thenucleic acid molecules when the host cell replicates.

[0202] The invention provides vectors for the maintenance (cloningvectors) or vectors for expression (expression vectors) of the nucleicacid molecules. The vectors can function in procaryotic or eukaryoticcells or in both (shuttle vectors).

[0203] Expression vectors contain cis-acting regulatory regions that areoperably linked in the vector to the nucleic acid molecules such thattranscription of the nucleic acid molecules is allowed in a host cell.The nucleic acid molecules can be introduced into the host cell with aseparate nucleic acid molecule capable of affecting transcription. Thus,the second nucleic acid molecule may provide a trans-acting factorinteracting with the cis-regulatory control region to allowtranscription of the nucleic acid molecules from the vector.Alternatively, a trans-acting factor may be supplied by the host cell.Finally, a trans-acting factor can be produced from the vector itself.It is understood, however, that in some embodiments, transcriptionand/or translation of the nucleic acid molecules can occur in acell-free system.

[0204] The regulatory sequence to which the nucleic acid moleculesdescribed herein can be operably linked include promoters for directingmRNA transcription. These include, but are not limited to, the leftpromoter from bacteriophage λ, the lac, TRP, and TAC promoters from E.coli, the early and late promoters from SV40, the CMV immediate earlypromoter, the adenovirus early and late promoters, and retroviruslong-terminal repeats.

[0205] In addition to control regions that promote transcription,expression vectors may also include regions that modulate transcription,such as repressor binding sites and enhancers. Examples include the SV40enhancer, the cytomegalovirus immediate early enhancer, polyomaenhancer, adenovirus enhancers, and retrovirus LTR enhancers.

[0206] In addition to containing sites for transcription initiation andcontrol, expression vectors can also contain sequences necessary fortranscription termination and, in the transcribed region a ribosomebinding site for translation. Other regulatory control elements forexpression include initiation and termination codons as well aspolyadenylation signals. The person of ordinary skill in the art wouldbe aware of the numerous regulatory sequences that are useful inexpression vectors. Such regulatory sequences are described, forexample, in Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nded., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,(1989).

[0207] A variety of expression vectors can be used to express a nucleicacid molecule. Such vectors include chromosomal, episomal, andvirus-derived vectors, for example vectors derived from bacterialplasmids, from bacteriophage, from yeast episomes, from yeastchromosomal elements, including yeast artificial chromosomes, fromviruses such as baculoviruses, papovaviruses such as SV40, Vacciniaviruses, adenoviruses, poxviruses, pseudorabies viruses, andretroviruses. Vectors may also be derived from combinations of thesesources such as those derived from plasmid and bacteriophage geneticelements, e.g. cosmids and phagemids. Appropriate cloning and expressionvectors for prokaryotic and eukaryotic hosts are described in Sambrooket al., Molecular Cloning: A Laboratory Manual. 2nd ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., (1989).

[0208] The regulatory sequence may provide constitutive expression inone or more host cells (i.e. tissue specific) or may provide forinducible expression in one or more cell types such as by temperature,nutrient additive, or exogenous factor such as a hormone or otherligand. A variety of vectors providing for constitutive and inducibleexpression in prokaryotic and eukaryotic hosts are well known to thoseof ordinary skill in the art.

[0209] The nucleic acid molecules can be inserted into the vectornucleic acid by well-known methodology. Generally, the DNA sequence thatwill ultimately be expressed is joined to an expression vector bycleaving the DNA sequence and the expression vector with one or morerestriction enzymes and then ligating the fragments together. Proceduresfor restriction enzyme digestion and ligation are well known to those ofordinary skill in the art.

[0210] The vector containing the appropriate nucleic acid molecule canbe introduced into an appropriate host cell for propagation orexpression using well-known techniques. Bacterial cells include, but arenot limited to, E. coli, Streptomyces, and Salmonella typhimurium.Eukaryotic cells include, but are not limited to, yeast, insect cellssuch as Drosophila, animal cells such as COS and CHO cells, and plantcells.

[0211] As described herein, it may be desirable to express the peptideas a fusion protein. Accordingly, the invention provides fusion vectorsthat allow for the production of the peptides. Fusion vectors canincrease the expression of a recombinant protein, increase thesolubility of the recombinant protein, and aid in the purification ofthe protein by acting for example as a ligand for affinity purification.A proteolytic cleavage site may be introduced at the junction of thefusion moiety so that the desired peptide can ultimately be separatedfrom the fusion moiety. Proteolytic enzymes include, but are not limitedto, factor Xa, thrombin, and enterotransporter. Typical fusionexpression vectors include pGEX (Smith et al., Gene 67:31-40 (1988)),pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia,Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose Ebinding protein, or protein A, respectively, to the target recombinantprotein. Examples of suitable inducible non-fusion E. coli expressionvectors include pTrc (Amann et al., Gene 69:301-315 (1988)) and pET 11d(Studier et al., Gene Expression Technology: Methods in Enzymology185:60-89 (1990)).

[0212] Recombinant protein expression can be maximized in host bacteriaby providing a genetic background wherein the host cell has an impairedcapacity to proteolytically cleave the recombinant protein. (Gottesman,S., Gene Expression Technology. Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990) 119-128). Alternatively, the sequence ofthe nucleic acid molecule of interest can be altered to providepreferential codon usage for a specific host cell, for example E. coli.(Wada et al., Nucleic Acids Res. 20:2111-2118 (1992)).

[0213] The nucleic acid molecules can also be expressed by expressionvectors that are operative in yeast. Examples of vectors for expressionin yeast e.g., S. cerevisiae include pYepSec1 (Baldari, et al., EMBO J.6:229-234 (1987)), pMFa (Kurjan et al., Cell 30:933-943(1982)), pJRY88(Schultz et al., Gene 54:113-123 (1987)), and pYES2 (InvitrogenCorporation, San Diego, Calif.).

[0214] The nucleic acid molecules can also be expressed in insect cellsusing, for example, baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al., Mol. Cell Biol.3:2156-2165 (1983)) and the pVL series (Lucklow et al., Virology170:31-39 (1989)).

[0215] In certain embodiments of the invention, the nucleic acidmolecules described herein are expressed in mammalian cells usingmammalian expression vectors. Examples of mammalian expression vectorsinclude pCDM8 (Seed, B. Nature 329:840(1987)) and pMT2PC (Kaufman etal., EMBO J. 6:187-195 (1987)).

[0216] The expression vectors listed herein are provided by way ofexample only of the well-known vectors available to those of ordinaryskill in the art that would be useful to express the nucleic acidmolecules. The person of ordinary skill in the art would be aware ofother vectors suitable for maintenance propagation or expression of thenucleic acid molecules dscribed herein. These are found example inSambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: ALaboratory Manual. 2nd, ed, Cold Spring Harbor Laboratory, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

[0217] The invention also encompasses vectors in which the nucleic acidsequences described herein are cloned into the vector in reverseorientation, but operably linked to a regulatory sequence that permitstranscription of antisense RNA. Thus, an antisense transcript can beproduced to all, or to a portion, of the nucleic acid molecule sequencesdescribed herein, including both coding and non-coding regions.Expression of this antisense RNA is subject to each of the parametersdescribed above in relation to expression of the sense RNA (regulatorysequences, constitutive or inducible expression, tissue-specificexpression).

[0218] The invention also relates to recombinant host cells containingthe vectors described herein. Host cells therefore include prokaryoticcells, lower eukaryotic cells such as yeast, other eukaryotic cells suchas insect cells, and higher eukaryotic cells such as mammalian cells.

[0219] The recombinant host cells are prepared by introducing the vectorconstructs described herein into the cells by techniques readilyavailable to the person of ordinary skill in the art. These include, butare not limited to, calcium phosphate transfection,DEAE-dextran-mediated transfection, cationic lipid-mediatedtransfection, electroporation, transduction, infection, lipofection, andother techniques such as those found in Sambrook, et al. (MolecularCloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

[0220] Host cells can contain more than one vector. Thus, differentnucleotide sequences can be introduced on different vectors of the samecell. Similarly, the nucleic acid molecules can be introduced eitheralone or with other nucleic acid molecules that are not related to thenucleic acid molecules such as those providing trans-acting factors forexpression vectors. When more than one vector is introduced into a cell,the vectors can be introduced independently, co-introduced or joined tothe nucleic acid molecule vector.

[0221] In the case of bacteriophage and viral vectors, these can beintroduced into cells as packaged or encapsulated virus by standardprocedures for infection and transduction. Viral vectors can bereplication-competent or replication-defective. In the case in whichviral replication is defective replication will occur in host cellproviding functions that complement the defects.

[0222] Vectors generally include selectable markers that enable theselection of the subpopulation of cells that contain the recombinantvector constructs. The marker can be contained in the same vector thatcontains the nucleic acid molecules described herein or may be on aseparate vector. Markers include tetracycline or ampicillin-resistancegenes for prokaryotic host cells and dihydrofolate reductase or neomycinresistance for eukaryotic host cells. However, any marker that providesselection for a phenotypic trait will be effective.

[0223] While the mature proteins can be produced in bacteria, yeast,mammalian cells, and other cells under the control of the appropriateregulatory sequences, cell-free transcription and translation systemscan also be used to produce these proteins using RNA derived from theDNA constructs described herein.

[0224] Where secretion of the peptide is desired, which is difficult toachieve with multi-transmembrane domain containing proteins such astransporters, appropriate secretion signals are incorporated into thevector. The signal sequence can be endogenous to the peptides orheterologous to these peptides.

[0225] Where the peptide is not secreted into the medium, which istypically the case with transporters, the protein can be isolated fromthe host cell by standard disruption procedures, including freeze thaw,sonication, mechanical disruption, use of lysing agents and the like.The peptide can then be recovered and purified by well-knownpurification methods including ammonium sulfate precipitation, acidextraction, anion or cationic exchange chromatography, phosphocellulosechromatography, hydrophobic-interaction chromatography, affinitychromatography, hydroxylapatite chromatography, lectin chromatography,or high performance liquid chromatography.

[0226] It is also understood that depending upon the host cell inrecombinant production of the peptides described herein, the peptidescan have various glycosylation patterns, depending upon the cell, ormaybe non-glycosylated as when produced in bacteria. In addition, thepeptides may include an initial modified methionine in some cases as aresult of a host-mediated process.

[0227] Uses of Vectors and Host Cells

[0228] The recombinant host cells expressing the peptides describedherein have a variety of uses. First, the cells are useful for producinga transporter protein or peptide that can be further purified to producedesired amounts of transporter protein or fragments. Thus, host cellscontaining expression vectors are useful for peptide production.

[0229] Host cells are also useful for conducting cell-based assaysinvolving the transporter protein or transporter protein fragments, suchas those described above as well as other formats known in the art.Thus, a recombinant host cell expressing a native transporter protein isuseful for assaying compounds that stimulate or inhibit transporterprotein function.

[0230] Host cells are also useful for identifying transporter proteinmutants in which these functions are affected. If the mutants naturallyoccur and give rise to a pathology, host cells containing the mutationsare useful to assay compounds that have a desired effect on the mutanttransporter protein (for example, stimulating or inhibiting function)which may not be indicated by their effect on the native transporterprotein.

[0231] Genetically engineered host cells can be further used to producenon-human transgenic animals. A transgenic animal is preferably amammal, for example a rodent, such as a rat or mouse, in which one ormore of the cells of the animal include a transgene. A transgene isexogenous DNA that is integrated into the genome of a cell from which atransgenic animal develops and which remains in the genome of the matureanimal in one or more cell types or tissues of the transgenic animal.These animals are useful for studying the function of a transporterprotein and identifying and evaluating modulators of transporter proteinactivity. Other examples of transgenic animals include non-humanprimates, sheep, dogs, cows, goats, chickens, and amphibians.

[0232] A transgenic animal can be produced by introducing nucleic acidinto the male pronuclei of a fertilized oocyte, e.g., by microinjection,retroviral infection, and allowing the oocyte to develop in apseudopregnant female foster animal. Any of the transporter proteinnucleotide sequences can be introduced as a transgene into the genome ofa non-human animal, such as a mouse.

[0233] Any of the regulatory or other sequences useful in expressionvectors can form part of the transgenic sequence. This includes intronicsequences and polyadenylation signals, if not already included. Atissue-specific regulatory sequence(s) can be operably linked to thetransgene to direct expression of the transporter protein to particularcells.

[0234] Methods for generating transgenic animals via embryo manipulationand microinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No.4,873,191 by Wagner et al. and in Hogan, B., Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Similar methods are used for production of other transgenicanimals. A transgenic founder animal can be identified based upon thepresence of the transgene in its genome and/or expression of transgenicmRNA in tissues or cells of the animals. A transgenic founder animal canthen be used to breed additional animals carrying the transgene.Moreover, transgenic animals carrying a transgene can further be bred toother transgenic animals carrying other transgenes. A transgenic animalalso includes animals in which the entire animal or tissues in theanimal have been produced using the homologously recombinant host cellsdescribed herein.

[0235] In another embodiment, transgenic non-human animals can beproduced which contain selected systems that allow for regulatedexpression of the transgene. One example of such a system is thecre/loxP recombinase system of bacteriophage P1. For a description ofthe cre/loxP recombinase system, see, e.g., Lakso et al. PNAS89:6232-6236 (1992). Another example of a recombinase system is the FLPrecombinase system of S. cerevisiae (O'Gorman et al. Science251:1351-1355 (1991). If a cre/loxP recombinase system is used toregulate expression of the transgene, animals containing transgenesencoding both the Cre recombinase and a selected protein is required.Such animals can be provided through the construction of “double”transgenic animals, e.g., by mating two transgenic animals, onecontaining a transgene encoding a selected protein and the othercontaining a transgene encoding a recombinase.

[0236] Clones of the non-human transgenic animals described herein canalso be produced according to the methods described in Wilmut, I. et al.Nature 385:810-813 (1997) and PCT International Publication Nos. WO97/07668 and WO 97/07669. In brief, a cell, e.g., a somatic cell, fromthe transgenic animal can be isolated and induced to exit the growthcycle and enter G_(o) phase. The quiescent cell can then be fused, e.g.,through the use of electrical pulses, to an enucleated oocyte from ananimal of the same species from which the quiescent cell is isolated.The reconstructed oocyte is then cultured such that it develops tomorula or blastocyst and then transferred to pseudopregnant femalefoster animal. The offspring born of this female foster animal will be aclone of the animal from which the cell, e.g., the somatic cell, isisolated.

[0237] Transgenic animals containing recombinant cells that express thepeptides described herein are useful to conduct the assays describedherein in an in vivo context. Accordingly, the various physiologicalfactors that are present in vivo and that could effect ligand binding,transporter protein activation, and signal transduction, may not beevident from in vitro cell-free or cell-based assays. Accordingly, it isuseful to provide non-human transgenic animals to assay in vivotransporter protein function, including ligand interaction, the effectof specific mutant transporter proteins on transporter protein functionand ligand interaction, and the effect of chimeric transporter proteins.It is also possible to assess the effect of null mutations, that ismutations that substantially or completely eliminate one or moretransporter protein functions.

[0238] All publications and patents mentioned in the above specificationare herein incorporated by reference. Various modifications andvariations of the described method and system of the invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of theabove-described modes for carrying out the invention which are obviousto those skilled in the field of molecular biology or related fields areintended to be within the scope of the following claims.

1 5 1 1114 DNA Homo sapiens 1 acagaattcg cccttttggc ttcaatctcttcccccatgc tcgaaggtgc ggagctgtac 60 ttcaacgtgg accatggcta cctggagggcctggttcgag gatgcaaggc cagcctcctg 120 acccagcaag actatatcaa cctggtccagtgtgagaccc tagaagacct gaaaattcat 180 ctccagacta ctgattatgg taactttttggctaatcaca caaatcctct tactgtttcc 240 aaaattgaca ctgagatgag gaaaagactatgtggagaat ttgagtattt ccggaatcat 300 tccctggagc ccctcagcac atttctcacctatatgacgt gcagttatat gatagacaat 360 gtgattctgc tgatgaatgg tgcattgcagaaaaaatctg tgaaagaaat tctggggaag 420 tgccacccct tgggccgttt cacagaaatggaagctgtca acattgcaga gacaccttca 480 gatctcttta atgccattct gatcgaaacgccattagctc cattcttcca agactgcatg 540 tctgaaaatg ctctagatga actgaatattgaattgctac gcaataaact atacaagtct 600 taccttgagg cattctataa attctgtaagaatcatggtg atgtcacagc agaagttatg 660 tgtcccattc ttgagtttga ggccgacagacgtgctttta tcatcactct taactccttt 720 ggcactgaat tgagcaaaga agaccgagagaccctctatc caaccttcgg caaactctat 780 cctgaggggt tgcggctgtt ggctcaagcagaagactttg accagatgaa gaacgtagcg 840 gatcattacg gagtatacaa acctttatttgaagctgtag gtggcagtgg gggaaagaca 900 ttggaggacg tgttttacga gcgtgaggtacaaatgaatg tgctggcatt caacagacag 960 ttccactacg gtgtgtttta tgcatatgtaaagctgaagg aacaggaaat tagaaatatt 1020 gtgtggatag cagaatgtat ttcacagaggcatcgaacta aaatcaacag ttacattcca 1080 attttataac ccaagtaagg ttctcaaatgtaga 1114 2 350 PRT Homo sapiens 2 Met Leu Glu Gly Ala Glu Leu Tyr PheAsn Val Asp His Gly Tyr Leu 1 5 10 15 Glu Gly Leu Val Arg Gly Cys LysAla Ser Leu Leu Thr Gln Gln Asp 20 25 30 Tyr Ile Asn Leu Val Gln Cys GluThr Leu Glu Asp Leu Lys Ile His 35 40 45 Leu Gln Thr Thr Asp Tyr Gly AsnPhe Leu Ala Asn His Thr Asn Pro 50 55 60 Leu Thr Val Ser Lys Ile Asp ThrGlu Met Arg Lys Arg Leu Cys Gly 65 70 75 80 Glu Phe Glu Tyr Phe Arg AsnHis Ser Leu Glu Pro Leu Ser Thr Phe 85 90 95 Leu Thr Tyr Met Thr Cys SerTyr Met Ile Asp Asn Val Ile Leu Leu 100 105 110 Met Asn Gly Ala Leu GlnLys Lys Ser Val Lys Glu Ile Leu Gly Lys 115 120 125 Cys His Pro Leu GlyArg Phe Thr Glu Met Glu Ala Val Asn Ile Ala 130 135 140 Glu Thr Pro SerAsp Leu Phe Asn Ala Ile Leu Ile Glu Thr Pro Leu 145 150 155 160 Ala ProPhe Phe Gln Asp Cys Met Ser Glu Asn Ala Leu Asp Glu Leu 165 170 175 AsnIle Glu Leu Leu Arg Asn Lys Leu Tyr Lys Ser Tyr Leu Glu Ala 180 185 190Phe Tyr Lys Phe Cys Lys Asn His Gly Asp Val Thr Ala Glu Val Met 195 200205 Cys Pro Ile Leu Glu Phe Glu Ala Asp Arg Arg Ala Phe Ile Ile Thr 210215 220 Leu Asn Ser Phe Gly Thr Glu Leu Ser Lys Glu Asp Arg Glu Thr Leu225 230 235 240 Tyr Pro Thr Phe Gly Lys Leu Tyr Pro Glu Gly Leu Arg LeuLeu Ala 245 250 255 Gln Ala Glu Asp Phe Asp Gln Met Lys Asn Val Ala AspHis Tyr Gly 260 265 270 Val Tyr Lys Pro Leu Phe Glu Ala Val Gly Gly SerGly Gly Lys Thr 275 280 285 Leu Glu Asp Val Phe Tyr Glu Arg Glu Val GlnMet Asn Val Leu Ala 290 295 300 Phe Asn Arg Gln Phe His Tyr Gly Val PheTyr Ala Tyr Val Lys Leu 305 310 315 320 Lys Glu Gln Glu Ile Arg Asn IleVal Trp Ile Ala Glu Cys Ile Ser 325 330 335 Gln Arg His Arg Thr Lys IleAsn Ser Tyr Ile Pro Ile Leu 340 345 350 3 57699 DNA Homo sapiensmisc_feature (1)...(57699) n = A,T,C or G 3 tttttgcaat ttacaatataatggctataa acattacata cttttacttt gctttattaa 60 cattttctcc atctttttctcaagtccaga caattaacca aacagtgaat caagccctga 120 tttgtagcat ttgcccattttcatggtata aatacttcat tcctgccatg gtctgctgca 180 agctaccgtc attatgtcattaaacccgga gttgagaaga gatgcatgag agcatgtcat 240 tatatagaat atctaccttacaaatacaat agacataata acctcaagtg cataaataac 300 agtaaaacat agtaaaataatttaaaaaag ataagtgtgg tatttataat ttatttaagt 360 tgtattagtt tgaactctataaaactgtgg atatgtaacc atctttagac cacagaagtg 420 ataaataagt gactgattcaacataaaaca cactcaggat gtttaaggat ggtggaattg 480 tggtgggtgg atagagaatgataacaataa tggcagcctc tactatttat tgaatacttt 540 caaagtctgg gcactgtgaaaagctaggtt tgattctctg tgattttcaa caatcaaagc 600 agaatttaca ttgattcctttttatctagc ttctcccaaa cagttcggaa aaaccctgat 660 agagactgga gaatttctgggactttagtt ccaattaaag atttgacaca atatatatga 720 aacactttat atgcaaatagcattcatgat attttacata attttttaac taaatacaat 780 atatagcaga acaagcactccttttggagt atgaaactca acacaagtcc cagcttcatt 840 aaatacttac tgtattgccagctgtgcatt ttgagctgcc tcacagcttc tgagggctgg 900 ttccttattt gtgattttagtaataatact gtctgtgcag attcacattg cagaataaac 960 aggagattca agtaggaaaaagaaaataga gtgccttcta aacagtccaa ttttgcagag 1020 gtagtggctt attattatagaatcatagcc ttctgtatct gtttccagga ttgtttcctg 1080 ggctatgccc ttcgactggctgggaagcaa ctaccagcct ttggcaattt tagggcattt 1140 ctgaccattt gtcttacaaatgaactgtgt ggtagaatta ggttctcatg actcttcaaa 1200 ttgcctagtc acatttgatttacttatatt ataataataa tgataataat aattatcatt 1260 attattttga gacagagtctcactctgtcc tccaggctgg agtgcagtgg cacaatctta 1320 gcttactgca acttccgcctcccacgttca atcgattgtc gtgcctcagc ctcccgagta 1380 gctgggatta caggcatgcaccacaaagcc tggctaattt ttgtattttt ggtagagacg 1440 aggtttcacc gtgctggccaagctggtctc cacctcaggt gatctgcccc gctaagcctc 1500 cgcaagtgct gggattacaggtaagccatc atgccagcct aaattaaatt atttttatta 1560 aaaaccctct ctttgttcatattgatagtc ctcaaagaat tttggattag tgtaaattac 1620 agttgaaagc tcagctggtggtaggaatgt ttacaaaaca ttcaggagtt tgaagcctcc 1680 caatagtaaa gctgggaagggtcccatctg tgggagagta agaattgagc ctggatcctc 1740 ccctgactct catatctctttttcccaatc gtggtttgtt gcctgggttt acttgggtca 1800 tctggctgat tcattgttgctgtataaaca gggctgagtg tgctgtgttg aaagccactg 1860 aaaccagagg aaactagtcacaaaaaccct gactatcacc tgatagattg cttgtgctgc 1920 ctgataatta ctcgcacttttcccaggcta gtgcaaatct tcaggggccg tccaggacta 1980 cagagctgtt tcaccctaccttggcttcaa tctcttcccc catgctcgaa ggtgcggagc 2040 tgtacttcaa cgtggaccatggctacctgg agggcctggt tcgaggatgc aaggccagcc 2100 tcctgaccca gcaagactatatcaacctgg tccagtgtga gaccctagaa ggtaagtgta 2160 gctcttctca ccctttaaaaagaaaaaaaa aaaaatgaaa tgatgtccct ctccagaagc 2220 atggagaaaa aaagcccataatcatatggt ttgagagttc tgagcagatc cttattcctg 2280 cagtccagat ttctggggtccataaacttg aatgaggaaa aaaaaataaa tctctatttt 2340 taatctctaa gtgaactataacatctttca ttactaatgt aggcaacaaa tcacagtagt 2400 tgagataagt agtatctgtgacactgtcac caacaaaaat tcaatgtttt catatccctt 2460 taaagttctt gcagataccttgaaatgtca tgtatgtttt ctctattttg aagtaagata 2520 ccacactcat cctttattatttatttattt attgagatgg agtttcactc ttgttgcata 2580 ggctgaagta tagtggcgtgatcttggctc accacaacct ccgcctcccg ggttcaagca 2640 attctcctgc ctcagcctcccgagtaactg agattacagg tgcccaccac ctggcccgac 2700 taatttttgt actttttggagagacgggtt ttcaccatgt tggccaggct ggtcttgaac 2760 tcctgacctc aggtgatccgactgccttgg cctcccaaag tgctggaatt acaggcgtaa 2820 gccatcatgc ctgccagattcttatacatt ttaaagacat tttgacaact atatctaaaa 2880 atatttggtt tcctttataatcctgtgtat tttcattcta catatttata aacatttgga 2940 gaagggcttc accaaactgccaagggatct atgacaaaga aaaaatatat atatttaaaa 3000 ccttgttcta tagaattttattctatcaca tttctcagta tctattgatt tgagagtttg 3060 aagtctacct ttaactgaagttaaaaaaaa aaaaatcaag agctgttact ggtggggcca 3120 gccctggatt caaaagtcctggatttgact tctgtcatgt caccagtagg tgacctgtgg 3180 ctccaagcct ctgagtctcagtgttctcag ttataataaa taaatactca acaagtggaa 3240 attgtctcta aaccacattttttttttcaa aaccttacca aagattccac gtctgtaaca 3300 gtatttttat tattaaaaacagctacactc actgattttg aaacatactt acaaaaccca 3360 tataggtcaa taataaggtggtttaaagct tccagattat tctagaaaag gcaagaaagc 3420 actaagaata aagcctcctaggaagggtat taaaaggcaa aaacgaacca atgaaccttc 3480 taaaggtgac tttgcagtctcctcaattag ggctgaaggg tcctgaggcc acttgtttat 3540 agttctgacc agttaaagccagagttcaga cagaaccacc caagttacga caaaccctgc 3600 acttccatta gcattggaaacagcaagaac cttttagata actaattctg ttggctacaa 3660 ataattacag taaaaccccaaatcctaagt cacttcagca tattaaaata agagtttggt 3720 gactggagaa cgtcggtcacgtatagaaaa tctacataaa ataaagaaca cagggctgac 3780 taagtgtttc tgagaggagacattttaatg aattctattt agttttcagc caagctctta 3840 aatgagctaa ggaatgcaaatttaaatgca aggaatacat aaaatatctt tgtgatacca 3900 aaaaaaaaaa agaaggaaacaaaaaaaaga attcagagat aaatcaaggg aagccatatc 3960 taaagccata taaagaattctaggcctgga gaggtggctc atgcctgtaa aaccccgtgt 4020 ctactagaaa tagaaaaagttagttggatg tggtggcaga cacctgtaat cccagctact 4080 caggaggttg aggcaggagaatttcttgaa cctgggaggc agaggttgca gtgagctgag 4140 atcatgccac tgcactccagcctgggtgac acagtgaaat tctgtctcaa aaaaaaaaaa 4200 aaagaattct agaaaataggtcgggtatag tggctcatgc ctgtaatcct agcattttga 4260 aaggcctagg tgtgaggactgcttgaggcc aggagttcaa gaccagcctg agcaacacag 4320 caagacctcg tctctactaaaaattttgaa aattggctgg gcatggtggt gcatgcctgt 4380 agtcccagct gcttgggaggctgtggcagg aggatggctt gagcccaaaa ggttgaagct 4440 gcagtgagct gtaactgcaccactgtgctc cagcctgggc gacagagtga gaccctgtct 4500 caggaaaaaa aaaaaaaaaaaaaaaaagaa ttctagaaag taaaaagata aaaatcagga 4560 atttaaaaat tgtaccctatattttctgcc aaggtccctt ttaatcaaaa gacttgtcga 4620 gggaagggga tagatagaaggaagaagaaa tgagattaac agatctatcc tctacataga 4680 cagttagctg aatctggtatcttcattttc agtttgggac agaagaaaag gtttcaatcc 4740 ctctcaaaga cctcaagaataaagcttgtg tttagaaaaa catacataag caggcatatg 4800 cagaattttc catacagcgtcaggggatga gaaaccacct gaagccaccc cacctgtgct 4860 ctactgcttt attgtcattgtgtctcatga tttctaactc tctctcttcc caattctcaa 4920 ctacatttga atctgtgtgccaatctactg ttcagagact cccagaaata tcaagcatga 4980 atcatcaaac catgtttaggttatcatgac tgctgagaaa acaaatctaa acaagaattt 5040 catgtcctct gttagttgaggacactgggg cttagaatgg ctgtgaccac ctaacacgct 5100 attggtgatg gaggatggatagagctatct gtgtctcctt ttccacaaaa atatgccatt 5160 gcctcctgag tataaattgaagatatatga ataggtctgt gtggagttga tgtccactcc 5220 attttgaaac agaaaataatgactgctcta cctgattatt ttataggatg ttgagaaata 5280 aatagtgtgt gaacagcagttcaactgttg catctgtgaa tgtacttaag gagactcagt 5340 ctgccccaag ctagattcagagctgagaag aaatcaactt gttggaatag aatcctaaag 5400 gcttaaggat tttagtttacttttgcacaa aatatacacc atacaaacta tggataaaca 5460 acaggttaca ctgtgccattccaatactat atgttgaagc agggactttc tgtaaaagaa 5520 aaactaactt ggccaagattaaatttgctt tcagtctttt taagtcatct ggtctttaaa 5580 aggaaagtgt tctgatagcaacaaattgtt ttagaacaac ttgcaaggaa cataataagg 5640 ttttgggtaa atttagaagagagaaaaaga tcatccagca aagctttgaa aagtgttagt 5700 attcataatt gtgcttaattgtgtgcataa ttgggttaag tgtagtaaga aatacccatg 5760 tattctgagg cctgcccctgactaattgtg acttgattca gatctctgga tccactgtga 5820 ctcagtcttt cctaggtacagaagaaggaa attagttccc attgcctttg tcactggtat 5880 tgctttaaag ttgtatctataaagaaattt tagggtcttt aaaagtgact ttgaaccaaa 5940 acaataactt taaaagagcatattttaaaa actaacacta ctgcttgcat cagaattttt 6000 aaaaattaca aataagacttttggaaatat tcttccctcc tcaaaaaaga caaaatagta 6060 aatcaatact taaagtttagtagtaaataa tcttttctaa aacatatgac aacagtgtcc 6120 actggctctc aagatgtgggaggtcattct ggattaagtc tttttttttt ctttttgaga 6180 cggagtttag ctcctgtcacccaggctggt ctccaatggc acgatcttgg ctcactgcaa 6240 cctctgcctc ccaagttcaagtgattctcc tgcctcagcc tctcaagtgg ctagaattac 6300 aggcatgtgc caccatgcctggctagtttt gtattttttt tttttttttt ttagtagaga 6360 tgggatttca ccatgttggccaggctggtc tcaaactcct aaccccaggt gatccgcctg 6420 ccttggcctc ctaaagtgctgggattacag acgtgagctg ctggcctgga ttaagtcttt 6480 actttgtcct tgacacagtttgtttagaag ggaattaggg aagctgatta tatctcacta 6540 acaatggtgc ccccctgtcaaggggcttga gcttcagagc ttctccagca gtgtcttcag 6600 gtaggaatta attcaacaccatctagtaag tactttctag ctagcgccaa gaaagtaaag 6660 atgaacaaga tagagtccctgccctaaaaa ggatcctacc tggagaataa aaatattaga 6720 aaaatgagaa ccccttaaggatggcaattg ggtagtacct actgaaattc caaatgctta 6780 tacactttgt ccaggaattccaagtctcat agacaaactt acacacatgt gagatgacag 6840 ctatacaagg ctctttatgtggcatagttt tttgttgttg tttttgagac agagtctcac 6900 tctgtcaccc tgggctggcatgtaatggca caatctcggc tcactgcaac ctctgcctcc 6960 tgggttcaag tgattcttgtgcctctgtct cccgagtagc tgggactaca ggcgtgcacc 7020 acccatgcct ggctaattttttgtattttt agtagagatg gggtctcacc atgttggcca 7080 tgacaggtct caaactcctaacctcaagtg atccacccac ctcagcctcc caaagtgctg 7140 ggattacagg catgacccaccgcggctggc cttttatgtg gcatagtttg gaatagcaaa 7200 agatggaaac aaatcaagtgtccctctgca ggaaataagt tttcattttt taaaatttat 7260 ttttttaata gagacagggtcttatgttgc ccaggctact ctcaaactcc tgggctcaag 7320 caatcctccc accttggccttctaacatgc tgggattata ggcctgagcc accacaacca 7380 gccaggaata agttaaataagttatggaac attcacataa tggaatattc tgcatttaaa 7440 aggaaaaacg catgaggaagcactctaaaa ggagaaatat ggaaagatta ccaagataca 7500 ttaagtcaga aaagcaaggggaagaaccag gtgtggcatg gcactcttta gtaaaaagtg 7560 ggataaattt taaaaatatacttcctatat gattacctag ctacaaagaa actgtgaaag 7620 gattaaaaag aaactaaaaatagaggttac gtgaagctag gaacgagtgg cagggggtgg 7680 tgggtaggtg agtgacaagcatagggacct gtcactgttt acccatatat acacctatta 7740 cttattttta atttcaaaataacatatggg aggatgactg atttcaaaga gctttctctt 7800 tgcttcctta gacattatttgtacctctaa ttatagcact ttttctgcct tatattaaaa 7860 agacatatcc aattctctcctgtttaaaac acatacacac acacacacac acacacacac 7920 acatcaagag cttccatcaggcaagataaa tgataaatga cagcttaaaa ggctgatcca 7980 ccttgtaagc ccttcgtcagaatgttttgc cttttctaga gctgcacctt caatcacaaa 8040 ttccatccct tatcccatgttcctggaaag cgaccatctc cctttaaagt cttcatttaa 8100 gtcacttcct ctgtaagccccttcctacag aatctatcat gggatggnnn nnnnnnnnnn 8160 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 8220 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 8280 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 8340 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 8400 nnnnnnnnnn nnnnnnnnnncctcagtctc ccgagtagct gggactacag gcacccacca 8460 ccacgcccgg ctaattttttatatttttag tagagatggg gtttcaccgt gttagcttgg 8520 atagtctcga tctcctgacctcgtgatctg cccatctcgg cctcccaaag tgctgggatt 8580 atagacgtga gccactgtgcctggctgcct gtaaacttct taaaggcaga aagtacacct 8640 taaacttttc tctctcctacctccctaagc ctaagagtgt attttgcatg tagtaagaat 8700 ctgaaaatac acactggaaatatctacata ttgggttcaa gtcctggatc ttctgcttta 8760 ggttctggga ttctaagcaagtgcttaacc tttaacatgg tgcttttttt ttttattttt 8820 atttttttat tttgagacagggtctcattc tgttgcccaa gccagggaat gcagtagtgg 8880 gtagcgtgat catggctcaatgcagcctca atctcctagg ctcaagtgat cctcccacct 8940 tagcctttca agtacctgggaccacaggtg catgtcacca agcctggcta acttttttgt 9000 catttgcaaa gacaaggtctcactttgttg cccaggctgg tcttgaactc ctggcctcaa 9060 acaatcctcc tgcttcagcctcccaaaatg ctgggattac aagcgtgagc actgataagt 9120 gctttcttat ctcatctttagatggtatct ccgtggccta actcccagtt cactgggagg 9180 atccaaaata tctgaagatgttttgtaaat tgtaaatctt aacaatacac ccctgcactg 9240 tagtttacat acaacaagaatttgttttcc ctcttctgct agactttgga attcctgaaa 9300 tagttctttc cattttatggttttggatag tggcatttgg atatgaatcc atttagttta 9360 ttctgttctt cagagagcaccagttttgaa aatatttatt ttggtaagta aaaattaaaa 9420 tgaagatttt caaccctagtaagatttact ggactgtata atgaagttct gtggggccac 9480 tgctacatgg atcttaagttttaatcacaa attaatattt gaggctcatt tatgtggcaa 9540 aaaaatgcat ttggagctgtggagagtaca aaaaagaaca ggctggatgc agtggctcat 9600 gtttgtaatc ctagcactttgggaggccga ggcaagagga ttgctagagg ccaggagttc 9660 aaggttgcag tgagctatgattatgccacc gtactttagc ctgggtgaca cagcaagaat 9720 ctgtctaaaa aaaaaatgacaccatttact acctagacaa ctgtgtagtt gataggatgt 9780 cacaaataag taaaatgagataaaaattca gaatccaaaa atggttcaaa cctattgata 9840 gcagatatac ttgaaggtgaaccaaatctt gaataataga ttgataaaag gccatgaaat 9900 ggggtttcag gaggagaagaattgagatgt caggacattc agcaagcaga taaaacaaat 9960 gaatttaaaa agggaaaaaataaaagcctg tggatgggga gtatatcagt taggctgcaa 10020 tactagaata cttggagagaaggctggaat tcaggctggg aaaactctga aaaaccttga 10080 aggtttgatt tgaagtttcaagatcctgac gctaacactg aatgaggaca agagtcaggt 10140 atgattctca gttaactacttgccagcgtg gaaaacagga acaagttact tctctgagcc 10200 tcagtttctt catctatgaaataggattat tgttaaagtt aagagattat gtatactaaa 10260 accattctgg caatggtaaaacgctctaca aattttataa aagttgtttt taatctgcct 10320 aaaacttttc agtggctgcaagctattttt aggatcatta cagtaccaaa acaattctta 10380 ggaaagatta aacttgaggcggggagcaca tgaggagaaa tgagcctggg aaaatcgatt 10440 caaagattgc tgcagtaatcgaagtgccag atgttattca aaacgggagt gagcagcaga 10500 aatcagggaa gaaatgcagggcatttggag taataaagca ccaaagagga ctttgaagtt 10560 tcaacaccct gagaacggcaatatattagc aaaaatggag aaagaagtgt ctggttgtac 10620 atcttccggt gtatttattaaaagtctgag taattaccag atttgatggt tgattccaat 10680 atgttagttg gaaaaattctggtttagagg ataaaagctg aattccttag caatttattt 10740 tggagatagg tgggtacaattgaaaaaaca atattttgtg gtggattctc tatgctggct 10800 ctttacctac ctgaatcatctttttttgtt tgtttgtttc ttttcttttt tgagacagga 10860 tctcactcta tttcctgagccagagtgcag tagcttgaac acagctcact ccagcctcca 10920 cctcccaggc tcaagcaattctcccacctc agcttcctga gtatggggac cacaggcatg 10980 tgccaccaca cccagctaactttttttttc ttttgtagag ttggggtttt gccatgttgc 11040 ccagtctagt ctgaaactcctggcctcaag caatcctccc accttggcct cccaaacccc 11100 ttggattaga ggcgtgagccactgtaccag acaaccttgt ttaatcccca aaacaacctg 11160 aaagcgtaga ctatatccattttgtagatg aggaaactga gttcggaaaa ttcagataat 11220 ttccctaagt tcagcagcagcagagtgtgg ggaaaggcag ggggctgctt ggatagaaca 11280 gtagatagtc acttctctttatttttagta gagacggggt ctccctatgt tacccagact 11340 ggtctcaaac tcctgggctcagcaaccctc ctgcctcggc ctccagagta gctggggtta 11400 caagcatcag ccaccacgcccagccaaaaa ggaattttct aaaaatagtc tgccaacaaa 11460 ttagtagaca ataaccatgaagtctaactt ccctatagcc aagtgatctc agataattcc 11520 cttcatgcag tgaccacaacccacagaaaa cgcatgttta tattatgact cagtgcatac 11580 attcaggcac acattcacactatgtgatag actctatttg ttaaaatggt gtaatctatt 11640 ttttttattc tattgctgtgtggcacctat taaagtgatt tagttggtag tgatcctaca 11700 gtttggaata tattctattttccttatttt aataaaaagg aaaaaacacc atttgccact 11760 ctacctttct agataaaaagtagtgtaatt ttcccaagtg tgctctgtgg gcggcccatt 11820 gttttttagt ggcttgggaggttctgtccc ttactgtttt caaagaaatc actttcatgt 11880 agtaaaggct tttaattcctacagtaaaga agcttgtgcc ttccaaattt attttaccat 11940 aaagtcttat tactcacacacctatttaga taagcattta gggaaatgta ggaaaaaaaa 12000 ttcagtgaac tcattttgaaaagtataaaa ctatgagtac tgttgttatt attgacagtc 12060 tatcatcatc ccagagtgcatttcagaccc tcagattctc ttggaatctg aaagcaagca 12120 tcccttggat cattttagtgctggggaaga gaggttcctt ctattccact tttcactgat 12180 tacagtatta taagattcctacagggtatg aaacactgag acatttaatt tcaggatgtc 12240 cctggcagct gaaaggtttgaaagttattt agcttgaaag aaacatatgt cacatgagca 12300 aaatatcatg gttttgaaagaaataccatt tgtgtcactt ctgttggcaa atattccttg 12360 gtgctgagaa ttcaaactgtcaagttgcat aaaaatctga aatgccaaaa tgaaagctaa 12420 ataaggcatg gcaacaaagtagaattgttt taagttaatt aaataggtaa ttgagcaaat 12480 aaattctgct gtcagatgaacagcatttac gatgcaaaat ctactccaga cactgaggaa 12540 gactgcaaga accacattgtccctaattgc aggtgcaccc tcccccagct accagagcaa 12600 tccattcata gctcgcagtggcttcctgtt atctcaggat aaacatggca taccaaaccc 12660 tccctaggac agttctgcttatttctttgg cttcaattct cacctcccct tctctaccac 12720 tcctttaaaa ttgacacataaaaattgcat atatttatca tatgcaacat gatgttttat 12780 tttattttat attttattttttgagacaga gtctgtcatt cctttgccca ggctggggtg 12840 cagtggcgca ttctcagctcactgcaactt cagcctccca gcttcaagta attctcatgc 12900 ctcagccttc tgagtagctgggattacagg cgcacaccac cacacccaac taatttttgt 12960 ctttttagta gagacggggtttcaccatgt tggccaggtt ggtcttgaac tccttacctc 13020 aagtgatcca cccgcctcagcctcccaaag tgctgggatt acaggcgtgg gccaccacgc 13080 ccagcctatg gtgttttaatatatgtatta attgtggtat caaccaaaat taagtttgtt 13140 gaggcagaaa tagttcagtaaaggtttatt ggaagccaaa tgtaaggact gatcaaggaa 13200 gacacagcaa caaagctgtgtgtgtcccag agtctgctgc gagttggaag gcttttaagg 13260 gaaagtttag aagaacagaggaggactctt catactagag ttgtcctttt cattgcaggt 13320 cacaatacag aggttacaatcactggctac agatgatata caggctaaaa tgtctacgtg 13380 caagacaatc agtaaacttcatgatgcaga aaccaatcac caaaacttga tgatttggaa 13440 acaaatcagt gtccttttcagtgccagtag gttatgtatt aatcagtaca ttaaaaattt 13500 gagggactca cggtaagattctttactctg ggataggaca ggacaggaca ggacaggact 13560 ggcatcataa gacctccgccaggcaggtaa atttggaagc ctgcccaatg tgacctacat 13620 gttatcaatc aagtgaattaacaagtatgt gaattcacat acttattttt tgtggggaga 13680 acacaaaatc tactctttacatgattttaa aaatacaata cattgttatt tactatagtc 13740 accattttgt accataggtctcttgaagtt attcctccta actgaatcta actgagtttt 13800 gtatcctttg acatctttccaacccctcac cctctcactt gtcccccact aagtcgtttt 13860 tttctttttt tttctttcttttttttttta aatgtagaga caaggtcttg ctttgtcacc 13920 caggctagag tacagtggcgagatcatagc tcactgaagc ctcgagcccc tgggttcaag 13980 agatcttccc acctctgcctcccaaagtgt tgggattaca ggcatgagcc accactgctg 14040 gcccactcct tgccctttgatgaccactac tttactctct acttctgagt tatcaaaatg 14100 aaagatttct ttctttttgaaggccgagca gtatcctatg gtatatttat accacctttc 14160 ctttgtccat tcatccatcaatgggcactt agcttgattc tatgtcttag ctattgtgag 14220 caatgttata atgaatgtaggagtacagat atctcttcaa catactgatt tcatttcctt 14280 tggatatata cccagtagtgagattgctag atcatagggg gttctatttt taatttattt 14340 ctctcccact taatacttcagcaacactga actgctttga gctccttgca cacagcatac 14400 ctgtactaag tcatcgaatgcctgcctctg ttcttaatct ttctcccctc ttcctccact 14460 cttacataat attctaaggctcatctcctt caagacactt tcctaggctt cttgctccta 14520 tctttatgga tctgggtctcattttctatg ttcccataaa tcaaggacct tatctttatc 14580 actaaattaa cacattgtttgaatcatttg tgactgcctc tttctttacc aaacagtggg 14640 tttccttggg aatgaggaatatttctggtc tatctttggg tctcctgtag ttggcagcac 14700 ataatagatg cttaataaatgtttgagtgg aagatccaat aagctccata tgtgtgctgg 14760 atgttgccaa ggacattttagatactcaga tatgcaataa cacttacatg ttgggttctg 14820 cataaggagt cacaggtaaaagatataact aagatagtgt aactgccttc tgggttaaca 14880 tgcttaagac tatataatatcctataatag gtgttatgct agaggtttta tcagagtact 14940 ttcaaattca gatgcatgagagtgaataat tctgcctgag ataattaaat ctttacagaa 15000 caagtgataa tctaattgaattagttggca ataaaataaa gttttaagtg actttgaagt 15060 caggaatacc tggcttggaatcacagttta tctacgtgag atttttaaat tttttaatct 15120 atgtgatctt gaacaagttactaacctctc tgagcttcag tgttcttatc tgtacaaagg 15180 catagaaatc atttttacctcataaaattg ccatgagtat taaatgagat aatacaagtt 15240 cagtacaata cctggtacctaataagcact caatcactgg taactattat tttaatgtta 15300 ccagtagggt aacattgaagtgtagattat aataagccaa tctgaaaagg ggtgggggaa 15360 gaaattctag agtggggttgggtcagcatt tattgaagta tggatgctag aaaatacctg 15420 gtttgttcag gaggaagtgacaatcaacaa agctgaaatt cagttcaatc caattacctg 15480 gcaaccaaaa tggatgacatttaaaggatg catggcatgg tctggtggaa acgatgctgg 15540 gttataagaa taaggggactaatctcctac ttcagtgctc attagataac tccaccattt 15600 agtcccactg aactcaattcttctctgtaa aaaaggatac tgaaacagag caccgatggc 15660 tgcccgtgcc acctgcccccacacccatgg tagctactac taatacattt atcttttcca 15720 tcaagttggg atggagcatttgtatcctca acagtttccc aggtagctat ctccaaaaga 15780 ctaaaacctg tttgccatctgtggattagg tgtttgttag gttttttttt ttttcagatc 15840 tgctaaataa taccagatttcttagtaatt gaaatatttt gtgagggcca ggcacagtgg 15900 ctcctgcctg taatcccagcactttgggag cctgaggtgg acagatcact tgaggtcagg 15960 agttcgagac cagcctggccaacatggtga aaccctgtct ctacttaaaa aaatacaaaa 16020 attagccagg tatagtggcacacgcctgtg gccccagtta cttgggaggc tgaagcagga 16080 aaattgcgcg aacccaagaggcagaggttg ccgtgagctg agatcgcacc acggcactcc 16140 tgcctgggct acagagggagactccctctc aaaacaaaag aaaaacacat tttctggtgt 16200 gctggattat ccatattcacataacatttt tgttgcttta cagatcaagt ctaaatcata 16260 tttagacctt atattaaaatctaataagat aatacatatg tatgtaacac aaaattagca 16320 tgaattcaac taatgttgaaaaagctattt gtgtatgttc aaaatttaac ctgaattggg 16380 gtttcattta atttcagacctgaaaattca tctccagact actgattatg gtaacttttt 16440 ggctaatcac acaaatcctcttactgtttc caaaattgac actgagatga ggaaaagact 16500 atgtggagaa tttgagtatttccggaatca ttccctggag cccctcagca catttctcac 16560 ctatatgacg taagtgatgacagaaagccc taataagccc caatgtactc gtgcggatga 16620 cccctaacaa ttacagggtgggtatcaagc cagagtgaga cttaagacct tttccattgt 16680 ttgtaagttt aaattcattcacattttagc aactgataag ctcatagagc cacatttttg 16740 cattagaaaa tggatttcattgatttataa acatgattat atcagcggtg gtggcctcca 16800 tctaaaggaa tcttttcagttttcatttaa aaaaaaaaag tgttgccagt gtttaaggtg 16860 ttagtactga aggtaaatgttatgtgacat tcttggatca cttttcaggg tggggttgac 16920 aatatctcaa agtattcataaggtcacata aaccactcct aattcccttt gtaacagtat 16980 aagtagcagt tgtgaaagttatatatgttt tatttttatc catcatctgt ggttccaatt 17040 gtgcaacaca tgagctcctcattctacaaa atatttgggg ccgggagctg cggctcatgc 17100 ctgtaatccc agcactttgggaggccaagg tgggtggatc acgaggtcag gaattcgaga 17160 ccagcctgac caatatggcgaaaccccatc tctactaaaa atacaaaaat tagccaggcg 17220 tggtggcact cccctgtagtcccagctact cgggaggttg aggcagaaga atcgcttaaa 17280 gccgggaggc ggaggttgcagtgaggtgag attgtgccac tgcactccag cgagggcgac 17340 agaatgagac tctatctcaaaaataaaata aattaaatta aaaaaattga actcccacat 17400 aatagtgaac tgcacttttatgacaccaat taataaattt tacttagtgc tcaaatggac 17460 catttgaaat aagcttatgccctttcagtt ctcctagctg aaaaactggg aacaaagaca 17520 cacaaataaa tgagctgtgtgttatgtaga taaaatgtag atgtaaagca caaagttctg 17580 aagactaggt ccaagcccaggtctggtgcc tggcactcag gcatcaatat atttgtggaa 17640 agaaaatatc aatgaaaaaatgctgcatag tataagccaa agattagtca aaggggaaca 17700 aaagaacagg acttcatgtcaaacactgtg ctcagtacta gaagtaggaa attagagaca 17760 tgttatacga atacaggaaactgggcgttg gctgtatgaa agcatggctt tgggagacaa 17820 tctaagattt ttagaggcagattccaagaa aatataataa gagaaagcct ggaaaaatag 17880 agggcagaga ctgcttctgaaagagcagag cagtttcagc ctctgtctgg ttgaacgata 17940 aaatgatggc gctgcctcaaggagcaggga atgaaaagaa tccgcttatc tgaccaggaa 18000 gatgatactc cctctatccttctattcttt ctttgtcctt ccgtatcatc catttttttt 18060 tttttttttt tttttgagactgagtctcat tctgtcgctc aggctggagt acaatggcgt 18120 gatctcggct cactgcaaccttcgcctccg tggttcaaga gattttcctg cctcagcctc 18180 ctaagtagct gggattgcaggcatgtgctg ccatgcccgg ctaatttttg tatttttagt 18240 agagatgggg gtttcaccgtgttgtctggg ctggtctcga gcttccaacc gcaggtaatc 18300 ctcccgcctc agcctcccaaagtgctggga ttacaggtgt gagccaccat gcccggcctc 18360 catcatatat tcttccactttacaaatgac agtatctctg cccttcagtc cagtgaacaa 18420 gaaaatggaa ctaagaatatccaaatatac ttgctgagct gaacaaagtt aagtggacct 18480 taacttctac acatgcctttaatttcaggg gaaaataata tgtcagtcag aaagatttct 18540 cacactatag cagatattccttggcatcta aatacggcaa acagaaaata ttccatgaaa 18600 acagacctgc attcctaggcctttggcaca aaggaaaacc aatgcaaatt tcctattatt 18660 gacagtgggc aagagcagaagtaaaggaag gctcttctgt ttggaatttc acatcgcatc 18720 atacaatctg aggatcaattttacttaatg aataatatct ttaagaaaaa aagaaaagta 18780 attaagggac atgattacaatctttcaaga gttcagaaaa gcataaaata aaaatatcac 18840 tctgtgtcta catttcctcttcttccccac tcagtttctc ttccaaaagt aaccacttct 18900 cagtttctta tgtgttattccagaaaaata cttacatgta cgtgtgtcta ttgctacaca 18960 ctctcccacc cacacatttcaaatttatac aaatggaatc atattccttc tgtacttttt 19020 tcccacttaa tatttcttagaaatatgctt gtgactgtca ttctttttat tttattattt 19080 atttctgttt ttacagacagagtctcactt attgccccgg ctggtctcaa accatcctct 19140 caccttggcc tcccaaagtgctgggattat agacgtgcac catcatgcct ggaaaacatc 19200 atacttttta aataactgcatagaattggc tatatgtgtc atgactccat ttaccaatca 19260 tttattgaca agtagctaaattatttttat tttttccgct tacaaaaaaa tttgcaatta 19320 gcttatttcc tacgagattagacgagatca ggcacgttca gggtggtatg gccgtagatc 19380 aattagctta tttctatgcaggtcttgaac ttatttctaa gctcagtttt tagcagtgga 19440 agtagtgagt caaaggaatgtgcatcttta tattttagta aatatttcta agttctcaac 19500 aaaggtagca ccaattttaaccccctacaa caacctgtgg aaagacctgt gtccttttac 19560 cattaccaac attggatattttctttttaa tttttgccag tccaataggt taaaaaaata 19620 gtatctcttt gcttcaatttatatttcttc aggagtgaaa ttgatcattt catatttatt 19680 atttatgtgg atttgatacaggacctaatt gtctattttt gttagagttt tcagcagttt 19740 ttaatgacat ataacagctatttgtaaatt aagaaagttt agcccttcat aatatatgtt 19800 gcaaaactat tttctttgtgttatatattt taatttttat aatgtttgtg gttttttgct 19860 gtggaaacat tgaacacttttaattagcta tactcatcaa tgttttcatt cattgccttc 19920 tgggtatttt attgtaccctgtttagaaag gccttcactc caaattcata aataaaccca 19980 cttgtctttt cttctatttatttgttgtta cacatgttgc ttcatttttt acatttaaaa 20040 ctttggtcca ttttagaatttattttcgtg taaagaatta gactacagat ctagctcaaa 20100 ttgttttccc aaatagctctccctttctac tccatttctg taagtaagca agtagaagaa 20160 aagaccatag gtcaaggaaggaaggaattg tggatatacc tgaggcatga ctttagtgcg 20220 gatgtttctt gtttgccatactcagtcctc ctatcagcgc tatgggttag gataattcct 20280 tccatgttac ttacaggcaagacacagatg ctgatgtttt ctaagaattc acaaatatac 20340 atacagctag tcaggagtagagattagata tggacttaca taggactcag ttcagggatt 20400 ctggaaaacc ttcccagaagggctgatgtt actgagcctg gaaatacggt agaatttggc 20460 aatacgggag aagccttagcatccgggtct gaaacatgta gatgggaagg ttgttccatg 20520 cgtgaggaac agttcagttttggctggagt gggtgtgcca tataggaagg aggaagtggg 20580 gaaacgaggc tggagacgcaaactccagtc agattatgga aagtctttga tgccaggcta 20640 aggaagttga gccttatcacatcagagtgt ttttcttttc tttctctttg ttttctttct 20700 ttctttcttt ctttttttttctttctttct tctttctttt tctttccttc cttccttctt 20760 tctttctttt ttttttttttttttttgacg gagtttcacc cttgttgtgt tgcccagact 20820 tgaacctccg cctcccgggttcaagtgatt ctcatgcctc agcctcagga gtagttggaa 20880 ttacaggcgc acgccaccacgcccagctaa tttttgtatt tttttttatt ttttttagta 20940 gagatagggt ttccccatgttggtcaggct ggtcttgaac tcctgacctc aagtgatcca 21000 cccgcctcag actttcaaagtgctgggatt acaggcgtga gccaccgcgc ctgtccgcag 21060 agtatttttc aaggttgtgtgtatatgtgg tgtattttgt tggtatctgc aaggcccttc 21120 ctaaaaagta aaaaccttaaggagaaaccc aaaatgtaaa agagatgaaa gaagatgcca 21180 ggattactca cagaacatattcctcctccc agtgatggtt gggaggaagt agaaagtgct 21240 taggctttgg gctgagtcagatccaaattc atacctaatc ttcaatagct ggcatgagag 21300 aggctctgca agcagtggtgcctcctgcaa aaatacctgg catggataat tatggaaaca 21360 gtaaagattc ctgagctgaaggacaaaatg gccctagttg tgtggaagga aaaataacaa 21420 tcaatatact agacaacatttacagaatat tacaatatgc cggggacttt attaggttgg 21480 aaagctaagt gctttacaaatattacctca tttaaccctc ctgacaagtc ttactcaatt 21540 cttaccatca ccattctaaggatgaggaac ctagaactta agtgcaataa tctgccctga 21600 tcacagatct aacgagcggtggagtcatga tttgaaatag ttgttagcag taggcttcat 21660 aaaattattt tgctcagattttagttgagt tcagaaaatc tgaccatgac agtgacagag 21720 ggcagcccaa ttttatctgaatcagaaagc tgccagggtt tgtactttgg gattgggaaa 21780 caaatctcat ataaaggcaatgtgcccaga atacactggg aaatgctcta gttctaaaaa 21840 cagctgtgag tcctttggtcaaaggttccg ctcactgatg atcccacaga tttcttgttt 21900 cctaatcata ggatcttttttttttttttt tttgagatgg agtcactctc acccaggcta 21960 tagtgcagtg gcgcaatctcggcttactgc aagctccacc ccccaggttc aagtaattct 22020 cctgcttcag ccttccaagtagctgggtct acaggtgcac accaccgtac ccagctaatt 22080 tttgtgtttt tagtagagatgaggtttcgc catgttagcc tggctggtct caaactcctg 22140 acctcaagtg atctacccacctcggtctcc caaagtgctg ggattacagg caggagccac 22200 tgcccctggc cctaatcatagaatcttaga catggaagga aacttagcat tcattaatta 22260 agcaagcatc cttatttcacaacaaactac tgtgttacaa atatactgtt tctaaaacaa 22320 aaataattac ttaaaattttaaatatttct agttataagc agtattttta aaaattatgt 22380 acaaaaatac agataatcacaaagcccagg gtggaatgct aacacaaaat tagaattgga 22440 aaggtggttt tggtagtatcagtattgtta ctaaaggata tattttaatt tatctactct 22500 tcctattggc tatacatgctttgaattagt gcttttagta tctctgtctg gactccctat 22560 tacctaatgg gcattgaatatttgttgaat caatatagaa tcctgcattc agcagtactt 22620 tacacggagc tagaaaaatatatccagata actcagcagt ttatatttta tatagcactt 22680 gaaaattagt tttttattgaccattatgtt aggggtaaaa ctggatgtca tgcctgcaga 22740 tacattttgt gaggccagctcagtgtttta aaaaataact tttaaaaaat acactttgag 22800 ccaggcacag tagctcatgtctgtaatccc agcactttgg gaggccaagg tggaaggatc 22860 acttgaggcc aggagtttgaaaagcctggt gaacatagtg agaccctgtc tctacaagag 22920 aaaaaattta aaaattagcccagtgtggtg gtacatacct gtagtcccag ttactgggga 22980 gtctgaggca ggaggattgcttgagtctag gagattgagg ctgcagagcc atggtcatac 23040 aactgtacct cagcttgagtgacagagtga gaccctgtct caaaaaaaac caaaaacaaa 23100 catacaaaaa acactttgaagtcaggtgtg gtggcttgca cctgtagtcc cagtttctca 23160 ggaggctgag gcaggaggatcatttaagtg caggagtttg attcaccagc ctgggcaaca 23220 cagcaagacc ctgtctctttaaataaataa attaaaaaaa ttttttgtca aggctgcatt 23280 cttccattag ccacagtctccaacactctt gtatttaagc tacttatcag acccctgaac 23340 atatttactc tgacttaaatgttttacctt cacctctaca caagaaccct gcaaaacagg 23400 tgtttatatc tccaattttaaaattacgga aatggagtgt caatgaatta atgcattttc 23460 caaagttaca aaggtaagagacatggaagg gattccattc caggtctatc agtctagcaa 23520 gataatgatc tcaccactgtcctcctagtt tataataagg caaacaaatc agaaatatct 23580 gtcggtcttc aaaaaccactcatattcttg acccctactt cagtagattc tgatctagtt 23640 ggccttggaa tggagcccatgtgtattttt gaaggcaccc aggttatact gaggtgcaca 23700 tctgatttaa aatgtttaatcagggggcct cagattgatt acttgaaggt gactttcaca 23760 aatacttcca tgagtttctgttgacagtta cctatacaga tagcattatc tccctgtcag 23820 agactagata gcaccaaagcctaggtcttc tgaatctcca tccatgatct gtcccatcat 23880 gtcatgtgat gtcttgtgactagcaaccct ggcagcattc agttgctgtc tgtagacctt 23940 agaggtttat tacaataagaaatgatatat ggccagacac agtggctcat gcctgtaatc 24000 ccagtacttt gggaggccgaggtgggcaga tcacttgagg tcaggagttt gagaccagcc 24060 tagccaaaat ggtgaaactctgtctttgct aaaaatacaa aaattagcca ggtgtggggg 24120 cgcatgcctg tagtcctagctacttaggag gctgaggcgt gagaattgct tgaacctggg 24180 aagtggaggt tgcagtgagctgagatcgtg ccactgcact ccagcctggg tgacagagca 24240 agactccacc atttcaaaagaaggaggagg aggaggagga ggaaggagga ggaggaggag 24300 gagaaggaga aagaaggaaaaggagaagga gaaggagaag gaggaaagaa aaaagaatat 24360 acaaatggtg gaaaggtatcccacagttct gaaaccattt ctcttatctc tcttccagta 24420 tatcttgcaa attcgagggtctctctagca cccatgaaat tccatcaaat gaagtaactt 24480 cctgtcctat tagaaaatgcccgagacaac gttatacaaa tgattaaatc tgttttatta 24540 gagacagtct aaatttaagttggtataata aatacaatct taagcattgc acattttatg 24600 agatataatt actgtacatgtgaaacttat tttgaaaatc tggaatggat atttttaaat 24660 acttaatagc aggaagatctgctggagggt ttgtatacta taattataca cagttgtgtg 24720 aaacaaagga atgaatgcaataacatttta acgaagagat catttaatat aggccttggt 24780 tgttttattt ttccttcttcctattgaaca ttttccatca gcatttcaac ttctcatctc 24840 ttttgaaacc aggcatctttttaaaaataa tctctcaagc attcaaagta tcccacctca 24900 gaggctgtaa aatttctgagtagaggatga ccaaggaaat gtcatcttcg gtacttaaac 24960 agtaatgagt aatcctaggttaaagtccta attctcagtt tactaagaat ctgacgttaa 25020 ggaccacaca ctgaagaaatctttaaagat tcattcctaa aggctaatta aagttttaaa 25080 atatcccaat gacacataataaaccattta cagcctttgt ggtgtgctgt acattagggt 25140 actgaacacc ttagtttacccaaagtgttt cccaggactc agaacttgta gtgttgatac 25200 caggaaagtc cttggaaaaccagatcattt gatcacccta tcaccatata gtcaaaaaat 25260 gaacaacata actctggctcactaagggct tacattctaa tgcagcaagc aagtccacaa 25320 atggtgtaaa agtaagacagaattaagcca cagctataag agaaggctga acattcaatg 25380 agaggaacct agaggactgaaacaatgaat tttgacttta aatgattctg tagagaaagt 25440 agaattttgg ccattgtttaaaattggcat agattgaaag aataagtgag gcatgaaagg 25500 attttgtgtt tggagatgacatggcatctc tgatattgct ggaacctaga catgagtagg 25560 gagaacagag acttgggcaagagagtaaag aggttgacca ggtgaagggc tttgtcacta 25620 agtctctaaa taaaagtcactacaggaagg ctttcagaaa atactatcaa ttccatgctt 25680 cttgtgtgct gcagacgataccccctcact taatggtcac cacgctgtaa gggggagaag 25740 tattttcccc ctgggtggccatgtgataga gtggttgaac acttccgcta cctctgactg 25800 tgtggccaaa ggcagtttgacatctaagca ttagtttctc atctagccaa tgggagagta 25860 cctgttctta ggattattgtgcatgtagta cttattcaat aactggtgat tgctattatt 25920 atgctgcttt cctggggcatacttaaaaat aaagtgtcca agtccttgta gctgctaagt 25980 aaaaagccta agatgtgatatcaagtcact ccaaatagca ttccaaaagt gatcattttt 26040 ttcttttttg cttatttgtatgtttttact ttttaactcc tgcttcgcaa agtgagggag 26100 ctgatcggat gtatatcttaggaagatgat tttgttagta ttataaaaga tgcatggagg 26160 ggatggaact taaagcagagaaacaataga ggaagctatt ttaaaagctt agactggaac 26220 tttgaatggt agagacaggaaggcggggaa tatgattaga ttcatttgag agagagattt 26280 aacaggattt agtatcttgatttatgaggc atgagggaaa gcagacaata attccctggg 26340 aattctaagt taaggaaagaaaggatttgc aagtaagata agtggatttt atttttagtt 26400 ctatcaaaga gactaggaagaccatggtaa attagaggaa attatagaag taaataaatc 26460 accctaaatc tcaccatcaattgacatatg cctatactct ttttctatac atttttaaag 26520 aactaccatt atcatcactttaattttgaa tcttggtttt tctctgtata ttatcagcat 26580 tttccccaca tctttaaatgagtaaatagc cttccatctg gtcatgaaca tagatatgtt 26640 gagttttgtg tctttctttccccaaacttt gcataatatt taatttgttg aattccagtt 26700 tcaacaatca aatttctaataatatgaaat tggctttttg acaccaccat gcccttgtga 26760 tcctgagtct ctggtcggaaaagttcttct gtggtcttat agattgcact gtcttaaagg 26820 ctttgcactc agagattcaatgggaatagt caaaggggag caagtgcagg gcaaacacat 26880 ccctactgta gttgctgataagaccttcag tgcttaaaac tctcaagcat tttgtgaatt 26940 gaaaaacact ggcgctattctgccaaagct atacttggtc tccctgtaac tgaatgatat 27000 tcatttaatc agcataacagtaagtgacta ctatgagtaa aggcttatgc gagactccaa 27060 gattagaaac gaattgaatatagactttgt tcattaacca atggaagagt tacttaagat 27120 gcagattcta agatcatgcttttaaaaatc atgatttact aggttttgga atgcagtcaa 27180 tgaatttgct cttctaaaagccctccaggc gaccctgata caggtgagaa attcttataa 27240 ccagtaggta gactctaaactgggtctaca atataccaac tgtaggaccc caaaaatgtc 27300 ccttagtcac tcaaagccttggtttcctca tctgtagtac agggatcgta attatataca 27360 atatagagtt gtgagtttaatttaaaaaca gaaccagata catagtttgt gaggcccggt 27420 gcaaaatata aagctccttgtttaaaatat gttcagtgat ttcaagagaa cagcagcaga 27480 gaattaaacc aagcattgagtcctctgtga ctacatagat cacactcccc tgaaactggc 27540 tgattgaaaa aaatgtatacttatacaata tataaatata cccacacatc catacacatg 27600 ctcaaaaaat gtagctaattttatttgtgt atttgagtat aatgttcacc tgtgtgagtg 27660 agaaaaggtg gcctgaaggaggtgatgccc aagctaaggc ttgaaaggct aagcgtttgt 27720 aggtggacat ggctggagagaggcaaacag cagagagaac agaaaacaca ggccccacca 27780 ctactgttgt tgggttgctgatgccacatg tcagcaaaat cagcgtctga ggttgaggat 27840 gttaaggtaa tggatgtgaaagagcttagg atgccatcgc tacaacaaca gttttgagtg 27900 ggatacaaat cagtaccaaggtgggcttgg gcaactgtat ggcaaggcat caatcaacca 27960 ccaggaggcg atattgatcccaataacctg ttttccaaaa cactactcct tccccagttg 28020 tttagtttaa gagatttgagactgtcccat aaccttcatt ttcaggatgg agactgaaat 28080 ccacacttat ccattgtcctagttttattt taacttgttt attttttaac tgaaaataag 28140 cacgtgtgtt taaaaaatgaaagaggtttt ttactaaaat gtattttgtg ctcactacac 28200 aagttagtac ctttataatcactttcctgt attaaattta atagattttc acagcaaccc 28260 tatggagtgg tactagtattgttggctttt tagaaataag acgatggatt gagtaaagac 28320 tatacttcag aattcatgtcacagagccca gaggtaacca ctcaaaactt tcatgcatat 28380 ctttctaaac tttttccttgtgaaaaatgt ttttttatat gtgcatgtgt ttatatttaa 28440 acacaaatat aaatatgcatagttgggctt attgtttgtt ttagaacagt gagatcatac 28500 atgtattgtc cacaatttgtccttttaaaa tgtaatctat tataattttt gcatcaatat 28560 ataaagatat attttatttaatatattttt aatttatgta ttatttactt aatttaattc 28620 attaacttat ttgtttttctgagacagagt ctcactctgt cacccaggct agagtgcagg 28680 tgtgtgatct tggctcactgcaacctccac ctcctgagtt ttagtgattc tcctgcctca 28740 gcctcctgag tagctgggattacaggcatg caccatcatg accagctaat ttttgtattt 28800 ttatagagac tgggttttgccatgttggca aggctggttc tcgaactcct gacctcaagt 28860 gatccacctg cctcagcctacccaaagtgc tgggattata ggcgtgaact accgtgctca 28920 gcctatatat atatatatatatatatatat ctttttgtat atagtataaa gttcaggtgt 28980 acatgtgcag gatgtgcaggtttgttactt aagtaaacgt gtgccttggt ggtttgctgc 29040 acagatcatc ccatcacctaggtattaagc ccagcatccg ttggctactc ttcctgatgc 29100 tctccctctc ccactcccctatagttattt attatctgta aataactatg tttgaaatga 29160 acatgtaaaa cgacttactcaaaagcacac agctctacag tggcagaaat tagtactgga 29220 atccaagcct tatgtcacactaaatatgta ttaaatattt gtgaatgaat gagctaacta 29280 gtccttatgt tcatcactgctactgccaat gtatatagaa aatttatttc accccaggat 29340 aaatataaat acagaaatttagaaagaaaa aagatatcac ctctagctgg agacaattga 29400 aaagcttcat gaaaaatatgatactagaat atggccttaa aggattggta aggaagggaa 29460 attttatacc tctggagaagggtaggaacc aagacattaa tgtagaaaag gacaagttaa 29520 gtttgaagaa tttttaatctaaacagttgg gttagaccaa gattgtagag aatcttaaat 29580 actgaagagg tttcttaaaattccagagca accaagggct tgggaaatgc aaagttatca 29640 gcattcttgt tctaaaatatgctattatgg caatgctgac ttgtaagtaa cttttcagta 29700 ttttattttg cttcaaggaaaaacaagtgt gacagcaaaa ttgtttgctt tttcaaatta 29760 ttttaatttt cacttcctctatttaaaaaa gggaagcttc agagaaggaa gaaatgttac 29820 caaaaatgtg aaattttaagagagagaaga atgtttatgg ggatattcta aattcagctg 29880 actaggaaat cacgcgatatgacagggttt ccccatgtaa gcatatgtgg ttagtgctgg 29940 attgaacaaa tccacagtataacaaaaaat gtttaccaat gtaatcaagg aaaactttca 30000 tttgaaaatt catacattgaaaaagtgtga aatatatttg gtgtactttt ataaattatt 30060 taattcctca atgaaactcgtaaatgaaag gtgcctttca cacacacaca ccgatccttt 30120 ccatgaacaa tgacccctgaaatgcttttt tttttttaat tgagacaggg tcttattctg 30180 tcacccaggc tgcagtgcagtggtgtgatc atggctcact gcaacctcag cctcctgagt 30240 agctggaact acaagcagtcaccaccacac ccagctaatt tctgtctttt ttgtagagag 30300 gcacctttgc catgttgcccaggctggtct caaactcctg agctcaagcg atccgcctgc 30360 cttggcttcc caaagtgctaagattacagg aatgagccac agctcccagc ctgaacatgc 30420 attatatgtc cctatccttgtacattctgt atgtataatt ggtacctagc caacatctca 30480 catagttctc tggtcacctaaaacaaaatg gttgtcatta agaattagat tacatgttgc 30540 agatgaagga tgtggatctggcctcctagg tagaaaggaa acctgtagca atcaaggcct 30600 gataaaagga tgtggctttctgggccaggc acagtggctc atgcctgtaa tctcagcact 30660 ttggaagcca tggtggaaagatcacttgag cccagcaatt agaggttgtg gtgagctatg 30720 attgcgccac ttgggtttggatgacaaaga cctgtctcta aaaaataaat aagctgggca 30780 gtggctcann nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 30840 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 30900 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnggacgct gaggcaggaa aaatcacttg 30960 aacctgggag gtggaggttgcagtgagcca agatcacatc attgcactcc gggcctgggc 31020 aacaagaagt gaaactccatctcaaaataa ataaataaac aaacaaacaa acaataaaag 31080 ggatgtagct cctgtgatcaagccaaatat tagtcacaat gtgtgtacac agtgaagctg 31140 ttgtcatcac caagttaaatgttctctcag gcccaaagga caaagttcag actgagtatt 31200 gaggtacctc catctagcaaaggggctcag gtctctttac aggaaagctg ttatcagttt 31260 tgacaaacag ttggcaagcaatgttttgag attactgatt acatttccaa agaagtaata 31320 ttgaactttt tgaagttcggataacttaaa ggaaacatca tataaaataa tgaccacaca 31380 aagacaactc tggggttacaagatacaaaa caagatggta ataggtttta tgaaataggt 31440 gtaaatcaga cttgagggctagtatcttaa cactattgag taatacgatt tatggtcttc 31500 ttttttggtg tcccagccttccaaatgact gtatcactca gcttcttgtc ttcacaccac 31560 aactgacatg ataatctcctattatctaag agcaataggt aaatttagaa agaactctct 31620 tagctaaaac taaatccaaaactacagaga tgaagaaagc tcaggcttac ctcactaaac 31680 ttttcaaatg ttttacattaaagtagatgt ggcttataat ttcctgcaaa gtccagttta 31740 caggaaaata tcttcttctaacagtgtatg tcctccttta tccaaggcac tctacgagtg 31800 ctttcatatg tgttactaagcctgatgcca accattcaaa gttgaatgaa tattttcatt 31860 tcaaaactga agaaactgaactcttagggt ttttaagttg cagagctggg cttaaatcag 31920 gtttatctgt ttcaaagcctgggctcctac tgtgacaact tctttatgat ttacaaactg 31980 ctttcctatg tgtaatttgatttagtgtgc acagcgaccc tgtgaagaca gtaattgtct 32040 acttcttctc accctgtgatgccaagtttg ttgaaaagaa taatctggcc aggcatggtg 32100 gctcatgcct gtaatcccagcacctttggg aggctgaggt gggaggactg cctgagccca 32160 ggagtttgag accagcctaggcaacacagg gagaccctgt ctctataaaa aaggaaaaaa 32220 attggccggg cacagtgtttcatgcctgta atccctgcac tttggggaag gccgaggcct 32280 gcagatcatc tgaggtcaggaattcaagac cagtctggtc aacatggtga aaccccatct 32340 ctaccaaaat tagctggatgtggtggtacg tgcctataat cccagctact caggaggctg 32400 aggcaggaga attgcttgaacccaggaggc agaggttgca gtgagccgag atcgtgcccc 32460 tgcactccag cctgggcaacagagtcagcc tctgtttcag gaaaaaaaaa aaagggaaaa 32520 aaattgacca ggcatgatggtgcatgcctg tagccgcagt tactttggag gctgaggtgg 32580 gaggattgct tgagcccaggaggtcgaggc tgcagtggtc tgtgattgta ccacagcact 32640 ccagcctggg tcacagagagagaccctgtc tcgaaaagaa aaaaaaaaaa aaaagaaaag 32700 aaaagaaaaa agaaaataataatctatact tctctatttt ctcacttatt tcctcatccc 32760 acttcggtct tatttccttccccatcaggt caccaaaagc tattctaata gctaagtcca 32820 atggatattt tttcagctgtttcctaattc gccatggcat ttactgttgt taccttgtcc 32880 cattcacttt gtgacaattctttgtgctct tttttaccat tttcactctt tctccttctt 32940 ccttgcaaaa atctcctttctatcccttaa gaaccaagga aataacacag tatggacttt 33000 aattaataac aatatgttaatatcgattca ttaattataa caaatgtgcc atactatttt 33060 aagatgttaa tgagaagaaactgggtatgg ggtatatgga aactctctgt accatcatct 33120 caattttttc tatgaatctaaaattattct aaaaattaaa ttatgttaaa aataatttta 33180 caaaacagag tttttctaatctcagtgtgt gcaatgctta agagacagac aggaaaaaca 33240 aaataaagaa tgagctaaatagaaaaagaa aacgacaggc ctgggagata gggcaaccat 33300 aagacaaatt atcacagaattcaagttcca ggaaggagga ggaaggtgtc ctggaggggt 33360 gtttacactc atcaaataccattgcaaatc ctggcagacg caccctgaga agcactccct 33420 ccccagagtg aagccctccttcctgctgct acttgaaagg aagaataata tgcccagctg 33480 cctgcctctg tcctcttccccatgtgccct atggggaagc ctcggagcat gtctgctggc 33540 cataacgacc ccccaccccagttcttcccc tttcaattct gttctcaagg tctgggttta 33600 tctccgtctc tgctcactctgactcagtac ttgtaagcac atcagaaatc ttaactctcc 33660 ccttctctac ctcagagacccttttggatt ttatcttaga gataaataac agtgtggcca 33720 gcggttcttt gtcatatagtgtccaatgtc ccttctgtcc ccttagatca gtgatcacca 33780 tcctttaatc atatacctatcattaacaca caagccnnnn nnnnnnnnnn nnnnnnnnnn 33840 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 33900 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 33960 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 34020 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 34080 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 34140 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 34200 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 34260 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 34320 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 34380 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 34440 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 34500 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 34560 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 34620 nnnnnnnnnn nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnngattata agatgaccac 34680 tttccagatc ctgagtgcctatatgtatat atactaaaac tacctaagag tacttgaagt 34740 cagaacactg tgctgggttccccttatcac ctccttatca caactgttct ttttccacca 34800 tccttttaca ccatactatgatgtatagcc ctgaggtata aaaagcttta gggcactaaa 34860 caaaccacag gaccatttgaagtgttagtg tgttgttttt ttaattgata aataacattt 34920 tccatattta tggggtccatgcaatttttt ttttacagtt atagaatgtg taatgatcag 34980 gtcagggtat ctgaggagtctatgactttg agtatttgtc attctgtgtt gggaaccatc 35040 agtctatttt gtaatatgtcatacattgtt gttaactata gtcactctgc tcttatgtct 35100 aactatagaa cttataccttttatctaact gtatatttat acctcttaat ctacttctcc 35160 tcctctcccc ctccctaccacccaccattc ctagcctcta ttatctgtca ttctactcat 35220 tacctttatg agatcaactttttttagttc ccacatatga gtgagtgcat atgatatttg 35280 gctttctgtg cctggtttattttacttaac atattgacct gcaattccat ccatgttgct 35340 gcaaatgaca tattattctttttatggcag aatagtattc tattgtgtat ctgtaccata 35400 ctttctttac ccattcatctgttgatggat gcttaggtta attccatatc tttgttatcg 35460 tgaatagtgc tgcaataaacatgtgagtgc aggtatccct ttgacatact gatttatttt 35520 ctttagtata gatacccaatagtggcattg ctggattgta tggtgtttct gtttttagtt 35580 ttttgaggta tcttcctactgttttccaca gtacttgtac caattgaaat ttctaccaat 35640 ggtgtataag aatctccttttttccacatc ctctctccag catattttat tgtttgtctt 35700 tttaatactg tccattctaaatggggtaag atgctatctt attgtggttg aagtattggc 35760 gtcttggagg acactaatgactaaaagata gagagaagaa gactgattgg ctaagaaact 35820 tgagacccag ggaatgacctggttttgagt ttctcggatt ttccttcggg ttgcctcata 35880 cactcctaac tggatgctagaaaatcttac agcccagaaa gccaatgggc acatacaaaa 35940 atgcccccaa ggaaaggctgttctctctag ccaaaggacc aggaaagggg cagcttcaca 36000 aacagaaagt cttttttttttttttttttt ttttttgaga tggagtctcg ctctgtcacc 36060 aggctggagt gcagtggcaccatctcggct cactgcaacc tccgcctcct gggttcaagt 36120 tattctcctg cctcagcctcccgagtagct aggactacag gcgcccacca ccacaccctg 36180 ctaatttttg tatttttagtagagacgagg tttcaccata ttggccaagc tggtcttgaa 36240 ctcctgacct tgtgatacacccacctcagc ctcccaaagt gctgggatta caggcgtgat 36300 tcaccgcgcc tggccacaagcagaaaatct tcagataata atggctctac tctaaccaag 36360 tgccatggga aaaactgcaaccccacccct gtcagcaaat gctcgatggg gacccagatg 36420 tctaccctta cccggctgtaagtggggtca gagagggctg agcgggaagc tgggcctttg 36480 accacctccc agcagtaatgagccctcttt ttagccttgg tgtcaataga ggccacacac 36540 agagtagtaa taaatcccagaaggagagga gaaaaaacga aaggttgaaa aagtattcaa 36600 agaaataatg gctgaaaattccccccattt ggcaaaagac acacaccgac tgattcaaga 36660 agctaagtga aaccccaaacaggaaaaatc caatgaaatc caggccaaga catgtcataa 36720 tcaaacttct gaaagaaaaaaagacaaaac aaatcttgaa agctatggga aatgacacct 36780 taccaataag agagaagcaatttgaaccat agcagatttc tcattaggca cctcatggag 36840 ccagaagcaa gcggtgtagtatttttcaag cactaaagac aagacaaaca aacagcattt 36900 tgtattcagt gaaaatccttcaggaatgaa ggggaaatca aggcattcta agatgaagga 36960 aagctaggag attttgtcaccagcagacct actctaaaag aatggctaaa cgaagctttt 37020 gaaacacaaa ggaaatgataaaaagagtaa tcttgaaaca atgggaaaaa agaaaggaca 37080 acagaaagga gaaaaatatgggtaaataca atataccctt tctcctcttg agttttctta 37140 attgtatttc acagttgaagcaaaaatcat tactctgtct gatgtggttc tcgatataca 37200 tagaggaaat ataagttataaacagggaag gaaaagacac ttaaaaagaa gtaaagtttt 37260 taaaactgca ctaaaattaggacagctact ctgaaaaata gtcttgcagt ttcttaaaaa 37320 actaaacata ttcttactatgccactcagc aactgcactt cagggcattt atccaagaat 37380 aataaaactt attttaatataaaaatcagc acacagatgt tcttagcagt tgttattcat 37440 aatagccaaa aactagaaaaaaaccacaaa tgtccatcaa taggtgaatg gttaaataca 37500 ctgtggtata tcaataccatggaatactac tcaccaataa aaaggaacaa actgtttaca 37560 tgcaacagct tgaatgtattacatgtatct caagaacact gagcagagtt aaaatagcca 37620 gtctcaaaat gtcacatattatctgctgcc agttatatat tattcagaaa atggcaaaat 37680 tatagagtgg gagaactgattcgcattttc tagacactgg gggtggtggg tgggaaaggg 37740 tgggtgtgac aatgaagaggtaataccctg gagagcttct tcactgaact gtgtttttct 37800 tagaaggtgt ttctctttttctatctcatg ccagagtgct tagtgaatga cagtcatttc 37860 tcagctattg ctaatgacactgaagtacta aaatttttat tcagagaaga gcaattcctc 37920 taacttacca aattatggctctataatatt tgacctctta gcaactgtta tagcagcaga 37980 atgtggtagt tatcatacagaaaatatatt atagttttct ttgagatcct taaataagct 38040 agggagcagg aggagaaagaatagctctaa gatcaaatgg aaatgttaaa atatcatctg 38100 gcttgagctc ttttatctgaacctactagg gagccagcac tttgatttta gggacccaac 38160 tctgctgctt gccagcactgtgatcttggg taaggtattt agtactgcta agcctcagtt 38220 ttccttaata atggttcttacagcattgag ttgttgtgaa gcttgagtga gagtgtgcat 38280 ataattactg ccacatagtaaaaacttcaa aaagaatcag catttaaaga ttttttaata 38340 tcctaaaatt tttgaaattgagtaatttgc tcatcacagt tttaggtgac tatgattttt 38400 acaggtgtca aaggacatttgtctagacca tgagtaggag aaaaattaac ctaactgaat 38460 gggtggctct gtcactacttatgaggataa ttatctaaaa cagaggtttg aactaggtaa 38520 acatcctggt gtctttttgttcaaaaatta ctcaggcaat gtcagcaatt atttgcttcc 38580 caaccactgc atgcatgaccccctggagat ctacaaagat tatagtctgg agaaagtaaa 38640 ctgtcttctg gttacattatgtcccctgtc accccaacag ccaatgataa ttatatacct 38700 ggtacctatg tgtgatggagatgagagagg ggacaggaaa gagacacccc tgcccctcat 38760 tgtgggccaa gccctgggttctccatgttt ctgaggaaca ctgcaagaac tggtattatt 38820 gttccatttt agaaacaaggccactcagta agagttgaaa taaaattgaa ccccatctat 38880 aaagcccttg ctcattgcaatatggagctt agcctgtcta ttagtaaaaa agtaaaaata 38940 aaaatcctga atacacacacatgcacacac tgtagaggca ggtctgggtg ttatatgcag 39000 gctgcatcct gctcacaccattgacgctgg tcgcccgtca acgttgctct aatagtcctc 39060 tgctaattag gaatgttcgcagctcagatg cttttacatt gcaggggggg ttgtggggaa 39120 caattaaatg tctggagtctgctaagcatt attaagtact agcaattgcc ctctgaggta 39180 attgcttttg agccataaagggacagttaa ataagcactt ccattttaat atctttttga 39240 agagataaaa aggattctaaaattccataa tgtctaaatt tggtactgct gttcaggagg 39300 tgaagaaact ttatagtcatgtgcttttta taaagggata attgcctgct tcaaaatctt 39360 ctctagttag aatttttggggttaagctct agagttccac aatgaaggag accccctttt 39420 ccaaccagaa acgaattccctgcccaggaa gtctgtaccc catgcagcca gactctggtg 39480 acaatgtttc tacgtgttcaagtaaatttt catccagtaa aaaggtatca tttttcaagt 39540 gtatctcaca aaggtggctaagcttcccat cactgtgaaa cctcgaggac taggtttgca 39600 ttgtcccttc ctccttctgatctgcgacct tgccttgtca actgcctcct ccccctcagc 39660 acttaaatat ctttacttctcttcctccca ccctaaaagg acacatatta aaactaaaaa 39720 tcgaaagcgt ccttggcccctgtatctcca tatatctggt tttctcttcc ttcaaggtca 39780 gcttgcagca tgaactccattctttaagcc tccatgtccc attaatttca gacacacaat 39840 ggccagtctt ccgccatcctcttctccctt tcccttgcac agacacactc cactgaaggc 39900 gttttcattg cattctctgatgacccctta gttgctgtac cctacagatg cctacttcca 39960 ggtctcaact tacatgtggactttgactct ccaatcactt cttagaactc tctagaactt 40020 cctaaaattc tctcccctgattctccttcc cccatctagt catccctctg aatttccttc 40080 aaggtcctta aaagctggggttccccagag ttcccaagaa agaactctct acatgcactc 40140 tacgatgtga tcctacttactcccaagact tcagcttcca cctttaaact aacccctcct 40200 gacccgctcg cccaaatgtctcgaatcggc agctgtcttc agggcacaca cttcttaggc 40260 ttacttgaca tctcctcttggttacctcct ggttaccttg aactcattcc atctaaaaca 40320 tctgcaccca ttgcaacctcctcttcccct aatgctctca tctcaataaa tggctctata 40380 acctaactgt caagcccagaaacctacaca tcaccccaaa aactaaaagc acataatgtg 40440 agggttcaga ggaagatttcagaagcagac tgcctgagtc tgagtctcag ctctcctact 40500 tgtcagcttt gtgtgtcatcatgggttgta ttttcacctc tctgggcctc tggtttctcg 40560 tctataaaat ggagataataatacgacata cctcacagtg ttgcagctat ggtttgaaaa 40620 gataactttt tgtaaagcacctcattgtat aggtgtttgt ttaccttttc tcctgtttta 40680 acatttacat aggtcataatattcccttca gtgtaagggc agtcagtatc ctccgttgcc 40740 cttaacattc aagttcctcatcttgtcttt cggggcccaa catgctcagc cccagcccca 40800 gctccagtgc tcggcccaggcaaaggaaac accctgagta tccttgtgtt cccatctctg 40860 gggcttttca aatgttctcccttctgcctt gaaatgtgct tttccccagt tcccagcccc 40920 ttttggctgg ccagcttctactaattcttc aagtttctac ttgactgtca tttctctagc 40980 aagtctaatt ccaggactggtttagcgtcc agctatgatt ctccacacca ccctataatt 41040 tattttgcat agcatgtatgccctattgtg atttgttgta aatgattctt tgtccatttc 41100 ctcacttaag ctgtattctttgtaaaggaa ggccctaggt ctcatttact tctcaacctc 41160 attgcttgta atatcttaggtgctgactac atgagtgaac caggacatgc acccagggta 41220 tcccgagcag agcttataccaaataaagaa atcacataaa aatgattaaa atatgaatta 41280 atatgaatta ataaactcggggatgtgagg aaggtggcct agaaaattca caatgtgatc 41340 tcatgcgagg acagaattacacagtatttt ttagaattaa attttgaatc ctcagtgata 41400 ggcttcattg cagtttcaaatatagtagtc agtttttaaa gtagagtaag atgctccagc 41460 tctgtgtttt tcaacttttcaagccctttg tccattctga taaaccaaac aagttcacac 41520 cctcaccccc caccatcacatttgagaatg actgtggagg ggcttcttcc gcatattccc 41580 accagccaag gttttatttagttagttgtt agaatattga atatgctttt tcatggccag 41640 gtgcaggcac catggctcatgcctataatt ccagaatttt gggaggccaa ggcgggcaga 41700 tcacttgacg tcaagagtttgagaccagcc tggccaacac agtgaaaccc tgtatctact 41760 aaaaatacaa aaattagctgggtatggtgg tgagagcttg taatcccagc tactcaggaa 41820 gctgaggcag gagaatcacttgaacccagg aggcggaggt tgcagtaagc caagattggg 41880 ccactgcact ccagtcagggtgacagagag agacttggtc tcaaaaaaaa aaaaaaaaag 41940 aatattgaat atgctttttcaatttatgac agtctcagag gtcatatatt tttctccaca 42000 tctcctaaac cccagtgtttaaaataaaaa gagactcatg tattttctga atgaaacagt 42060 gtgtacctaa agagtgtgtgttttttactt gtgggatgct tcttatcctt ttgcagctgt 42120 cctctaatga ttgagttgtcttctgaacca ggtgcagtta tatgatagac aatgtgattc 42180 tgctgatgaa tggtgcattgcagaaaaaat ctgtgaaaga aattctgggg aagtgccacc 42240 ccttgggccg tttcacagaaatggaagctg tcaacattgc agagacacct tcagatctct 42300 ttaatgccat tctgatcgaaacgccattag gtaggaacac ttaggtaatt ttgtagctgc 42360 ttgtgtatgt tatcacagtcattagaaaat gtactatttt tttcttccct gtggtccaaa 42420 agaaaatgta ctttgatttcacaatttttt tccatgaata cagtagcata ccatgcagat 42480 tcctggaagt ggcagagacattaaggaatt atctagttca gctccctcat ttttcagatc 42540 aggacaaatg aagcaacctgctcagatcac atgtgaacca tttgtagtag agaaggaact 42600 ggagctcatc tctcaaccttcaaacttttc actttatacc aggtcatagc aaataaataa 42660 tagcacttgt cattcttcaaaaaaaatgta attttctagc ttggttttac agaattagtt 42720 tcttgtcctt ccatttaaatttttgtctag ataaggctgc atataggaag cattttcttt 42780 caggaagttt aaaaatgtcatttttctgag atgctcacga gtatctgtca agtcatcaga 42840 agcaatgatg aaatgattgttaatttcatt ataatttggc actaacaaga gttgtgttca 42900 aaatgctaaa gagataattgtataagcatc attatctaat attgtcagga aatctggata 42960 gtagcaattc actattgatcgtttgtcaaa atagttgctg tttaagatag atattttaaa 43020 aagcaaaaaa aaaatatttgtgtcatcatt aagcacccga tactgagata gcataggctg 43080 atcgagagag gttgagactgcaatcagaca accatgagca tgaaaccagg ctctgcccgg 43140 tcccagcaag gtgactaggagcaattcaat tcaccccagt ttcctcattt tggaaataat 43200 cgtatctttt tataacagagttttaaaaat aaaacacact tttaagtaca actcaatgct 43260 tggcacatac taaagttgcaaataataaat attattatta gatagtgaag gaggagggac 43320 ttcaaaaaaa agccttaagtgaatagctcc aaggtgttgt gaagattaaa tgaaagtata 43380 ttcagtgtat agtgtagtgagtgaggttta gggcagcagt ccccaacctt tagggcaaca 43440 gggactggtt tcgtggaagacaacttttcc atgacaggag agaggggatg gtttcgggat 43500 gattcaaaca cattacatttattgcgcaca ttatttctgt aattattaca ctgtaatata 43560 taatgaaata atgatacaactcaccataat gtaaaaccag tgagagccct gagcttgttt 43620 tcctgcaact agacagtcccatctgtgggt gatgggagac agtgacagat catcaggcat 43680 tagactctca taagtagcacacaacctaga tccctcagat gcacagttca taatagggtt 43740 tgctctccta tgagaacctaatgctgccgt tgatctgaca ggagggggag ctcaggtggt 43800 aatgcaaaca gtggggagcagttgtaatta cagttgaagc cactctctgg cccactgttc 43860 acctcagctg tgtggctcagttactaacag gccacggact ggtgttggtc tgtggcctgg 43920 gggttgggga cccctggtttagggcttaca catgataaat gcttaaaaag taataactgc 43980 tttcaccctc attactattagccctattta gtggagtgta tggattgcaa ctctgcagtg 44040 ttcttgtcag gagtgagcagagggtggtgt gttcaggata cgttttctac agcttataat 44100 cttgcttaag attcattttttggtatggca atttctgctt tcagctccat tcttccaaga 44160 ctgcatgtct gaaaatgctctagatgaact gaatattgaa ttgctacgca ataaactata 44220 caaggtaatg gttttcccaaatattgtctt tttcttgatt acacaatgta ttgtgactag 44280 agttctctat taaaacttactttactcatt aaccaacttc tgcaattatg aaactgtgca 44340 tatttggaat atagcttttatagttctggc ctgtcctcta gttaaatgca tgcatgtcag 44400 cttcccctag ccagcaagatatttgaaagg aaataacatg ccctattctt ccatattcct 44460 agctccttgc atggtacctggaacataaaa ggtgtttgga tgatggtagg tgaagaaatg 44520 gggtgaacaa tatcaatccatttaaagcaa ccagcataca tagcaaacct tctgctatgt 44580 agtgttcatc tctgtcctcaatcctgttag tatccctgaa tctcacagct aatctgccca 44640 cacaatttta atctacagtgtgcagcatac cagaatgaac aatttgtgta cactgcatca 44700 taggtaccac tttgcataatgtgactgtct ggactgtgct gccaccattt gagtagctgt 44760 atatacttta ggctgagcaaaatctgtcca tttttagtca aataaccata gcctctttgg 44820 acactcacac acagtaactgaacatatatg gcatgctgtg ttcaaacatg tggtcctttc 44880 aaaagcattg tattgcaggcagtgttcctt ttggtaaagt gacttatggg cctagaggag 44940 gcatccttga gatataactaacagaaggca ccgttctaag caatttatag gccttaactc 45000 actaatccac ctggcaaaaatatgatgttt atgctatctc cattcctcag aggagaaaac 45060 tgaggcttgg agagccaaggtttctttgga agaagtcagt tagctaaaac gtattagagt 45120 tgggatccaa actcaggcagtctgacccta cagcctttcc catatagctc catgctcaaa 45180 gccatcccaa atgtgcctctacaagagact aaatgtcagc atagggctaa atctaaaatt 45240 attcactgat ctcagaaaagaacttggcac tcttgttaaa atagcaagaa ttaaccatat 45300 gaaataatcc aaaaccatgcaaaaaaagtg cttgttaagt aattataagt tatgataaat 45360 gcagttttgt ccttttctggaattcttttt tagactgtgc aacatgctgc aatgtgaaag 45420 cttctaccaa atttattataaaatatcttc ctatgggaga tgtaattcag attccattaa 45480 agcatttaat aaaccatgaaaagctgatct agacattttc aaaaggaata tatattcatt 45540 atatcacttt atgatctttcttctttttaa gtcttacctt gaggcattct ataaattctg 45600 taagaatcat ggtgatgtcacagcagaagt tatgtgtccc attcttgagg taagaaagag 45660 tccttgaatt ttgttatgctaaataaggtg cttacatatt tttattgttt taaccttact 45720 tatatttcct tatctttgaattttttttta atctagaagt aagacaagca gtacatttta 45780 ttatacatat gatttgatatatttgattaa gagttgtatt ttccaactct taagttggaa 45840 tatggaagtt ttctacatggaaaagttagc ttgggaatta gaaaataata atgtttacac 45900 agatttggtt atgattcattttaatgtagt atgtaaaaaa tagattgtaa acaataacaa 45960 atttagtata ctgttgttactggaaaaggg tcccaatcca gaccccaagt agagggttct 46020 tggatctcag caagaaagaatttgggtcca tagagtaaag tgaaagcaag tttattaaga 46080 aagtaaagga gtaaaagcatggctactcca taggcagagc agccccaagg gctgctggtt 46140 acctatttgt atggttatttcttgattata tgctgaacaa ggggtggatt attcatgtct 46200 ccccttttta gaccacatagggtaacttcc tgacattgcc atggcatttg taaactgtca 46260 tggtgctggt gggagtgtagcagtgaggac gaccatggtc actctcagtg ccatcttggt 46320 tttggtgggg tttagccagcttctttactg caacctgttt tatcagcaaa gtgtttatga 46380 cctgtatctt gtgctaacctcctatctcat cctgtgactt agaatgccta accgtctagg 46440 aatgcagccc agtaggtctcagccttattt tacccagccc ctattcaaga tggagttgct 46500 ctggttcaaa tgcctctgacactatgagca atggatggac tatagacact ccataatcac 46560 ccaaattccc tctggtgagggaacattggg cagctgggaa cccccacaac ttggcatcaa 46620 aagtccacta tagcttcatccacagggaca ttgtgcctta caaatcctaa aataaaggct 46680 gccccaagca acccatgaacattccagctt ttggcaggaa gactcatttg ttatgtgatt 46740 cacagttggt aagctcatttaattatgtgt caaacaccac cattggtatc tacctctcta 46800 gctgatgtta taacttaattcctcttaagg accatagatt tcatctatgc ttaggagtcc 46860 tcttgcataa gaatgttgacctgcatatgc atgggagatt cagggcagag atcagaagga 46920 tttgtggggc atcctgcacataatttgcac tgagtatttt acctccattc ctcttgctag 46980 taaataattt cctaattcacttaaaaagaa aaaatatcac tttgggcttt tgtaagccct 47040 tactttgccc tgtgaaactaaattttatct aaagaatatt tttcatcatt acattatctg 47100 gtacagtgac ttcactggtgggtggaattc tgatttcaag aaaagcctaa taagcattac 47160 ccttcaggta aaaagaaaaaaattaacttc atttgcaagt atttcaacct aatgctctga 47220 aactgcatat gataaaactagctcccctca aaacatctaa tcattaaaat tttacttggt 47280 tgatgacttc tgatatagaaaaaggaaaca aaaaagtttt atagttgaag atattttgct 47340 attccttatt gagattttgtttgtttgttt tgatttttga gacagggtct cactctgttg 47400 cccaaactgg aggagtgcagtggcatgatc atggctcact gcagcctcga cctcttgggc 47460 tcaaatgatc ctcccacctcagcctcctga gtagctggaa ccacatgcac atgccaccat 47520 gcccagctaa gtgttttattttttaaaaaa gtttgaaaag accaggtctt ggctgggcat 47580 ggtggctcac acctataatcccagcacttt gggaagccaa gctgggtgag cacctgaggt 47640 tgggagttcg agaccagcctggccaacatg gtgaaaccct gtctctacta acaatacaaa 47700 aaaaaaaaaa ttagctgaatgtggtggtgc acatctgtaa tcccagcttc ttgtgaggct 47760 gaggcacagg aggtgtgaaggttgaacctg ggaggtggag gttgcagtga gccgagatcg 47820 tgccactgct ctccagctgggcaacagagt gagactcaga aagaaagaga gaaagagaga 47880 aagaaagaaa gaaaagaaagaaagaaagaa agaaagaaag aaagaaagaa agagagagag 47940 agagagagag agagaaagaaagaaagaaag aaagaaagaa agaaagaaag aaagaaagaa 48000 agaaagaaaa gaaagaaggaaagaaggaaa gaaggaaaga aggaaagaag gaaggaagga 48060 aggaaggaag gaaagaaaagaaaagaaaag aaaagaaaag aaaagaaaag aaaagccgag 48120 gtatctctat gttgtccaagcctgaaattc ttaaaataaa gcctaaatag ttgtgaagtg 48180 aaaatcttat cactaaagatctccaaaaaa tattcttgtt cttatctata ttatacagaa 48240 agtggatatc gaataaataaataacatttt gaggcatagt aaatgacaga atgaaaaata 48300 gtctctctca gtcatgctaatcatgcaaca tcattaactg gtgaccctat gaagccctat 48360 cctttctttt cataaattcaactactagcc atactataag aatccatatt atctatggct 48420 caaaagtaga atttctactcagcataattc ttgtggtttg gaaatggttt gcttacttta 48480 tacccatgaa atatgtgaaataaaagttat atcaagactc ataaccttat caaaggtata 48540 caatagcact attaatagctaacacttact acgtgctaga tgctatcttg agtgctttac 48600 atacatgaat tcacttagacttcaaaacca cacagaatag gtagtctagt atctattttt 48660 caggaggagc aactgaggcacagagaaggc taataactta cccgacgtct cacagctagt 48720 aagtcaggat tggaatgcaagcagttggct cacgtactta tgtgcttatc cactctgcca 48780 tagtgcctct gcattaaattcaccattcac aattctttta tataaacgtg agtgctaatt 48840 cataagcaag atcatattgtgtactataaa aattggtaaa taggtaaggt aagaattaca 48900 tctttgaata ggtaagaaataggtaagaat tatggctttg taatccttgg taatgtaaaa 48960 aaaggatagt atcataaaaatgcttatcac tgggatgatg tgatcaaact acgatatgcc 49020 cagtgttctc attgtacttttccaatcttc atggtatcag atgagattag tgtttgtttg 49080 tttgtttcaa acagcattaaaaaaaaatcc agctaggcat ggtagtttat gcctgtaatc 49140 ccagcacttt gggagacctatgcagaagaa tcgcttgaag ccaggagttt gagaccagcc 49200 tgggcaacat agcaaaatcctacctctaca aaaaatacag aaattaaccg ggcatggtgg 49260 aacacaccta tagtcctggctactcaggag gctgaggtgg gaagatcgcc tcagcccaga 49320 aattccaggt tacagtgagccataagccac ctcattccag gctgggtgac ggagtgagat 49380 cctgtctcta agaaataaaaataaaaatcc attgaacagt ttttatttat acggaaatat 49440 cagctacaca gggcatgaggttataacatt tataattata tgatcaaagg gtgtttgtca 49500 gctttacata aatactttcgatcatgtaat tacctccctt ttacttttgt aactataaag 49560 cacttactgt ttataggttccatgtatatt ctcaattact ctcatttggc cagagacttc 49620 aagagactta atatggtagaagagaccatt aacctcagct tacatgtgaa aatttgacct 49680 aattacaaga ttataacttcatatgaaaaa aaaagaaatt gacttatgtt caaggcaaat 49740 gcactgtagt ataatatttgccttttactc ctactcttca atcttatggt taatagggca 49800 gtgaaaaaca atcataaaatatgtttgttt gatctctact ggtcagtgca tgtgctgata 49860 aggctgggct ccattccagttcacttcact cttttccatt gccactgaca tctccgccca 49920 ctgcctcctg ccaggtgcacccttcttccc atgaggaact ggagagaatg tgtgacccca 49980 tcggtctgag tttatgatgtgcaaccgata gtgattttga gcaagtaaac agttcattgc 50040 tatctcttga aataacaaaaaaaataaggt ctagttttaa catgagtaag tttttatagg 50100 gaaaagtaaa aggaaagtcacgggactgcc aacatacacc ataccacaca gcgtgccaac 50160 tctctgcaag aaacaggaactaagtcacac taacaccagc ccattcagtc tcctctaatc 50220 aaatgcgtgt caaactctgcttagggatgg tcaaagaaca gtccttttcc aattctaaaa 50280 cgatgggtgt tttcaccataggataataca cacacttacc caaaagagcc tctagactag 50340 aagagtaagg tagatacatagtcagttatg ataaaaggaa aatggaatgt aatcagggtt 50400 caaagggaat catatccaattagagaagaa atggaagggc ttgttatgaa aaagatgaca 50460 tttgatggga caggagcgcagggaggattt cagcatgcag agttatggaa cagagcacat 50520 gcacggtagg ctaagggaagaagtggttac aaggacaagg aggtagcgag aagatggagg 50580 gctcagataa cctactgtatcagccaaagc cctttcttct tatattcata ttctctcccc 50640 agccagctgt ccttcccacttctccaattg ctttcccaat gattgagcca tgacaattcc 50700 tccaaatcat cctgctttgtctcctgcagc tccctcttct ggcctggact ctgctgtttg 50760 ccagtctgca atgctgcctacgttgcccta attctcttcg taagatcatg acattttaga 50820 atcagagagc acattaaagaacatctgtga caatcacctc cattgttgct ctagttgccc 50880 taatttttgt tttaagttttcttattgcca cgaaatcttc ttcttccatt tcttctctta 50940 tccctagaag gtctaaaattactgacagga ctggttatat tcagtagcca gagacagctg 51000 cccttgcttc cacataaagccactccctac ttttcagctc acacataatc tccatgagag 51060 caatcaacag ggtcagatggaactaactct cactctcctg atagaaaaca cctgttctta 51120 cataggattt ccagtgaaatctgttgcaag ttgggttttg ttataggtta accagtcaaa 51180 agctgattag aggatagcatacagcaattc ttttcagctt tcacgtgcat atagatagcc 51240 tgggaatcct atgaaaatgcagattctggt tgagtagggt tggactgcgg cctgagactc 51300 tgcacttcta atgagtttccaggtgacgtc aaatctactg agccataaaa tattgctttg 51360 aatagcaaca ttatggagaatgttggagaa tttcaaagta gtaggatcac acatcactct 51420 gttggataca gtaaatacttaagactttct tatgtgtcag gcactggcta ttcaaaaatg 51480 aacaaaaggc tgggcgtggtggctcatgcc tataatccta acccttagaa ggcccaggac 51540 agaggctttc ttgagcctaggagttcaaga ccaacctggg cattatggca aaatcccgtc 51600 tctacaagaa aaattacctgggcatggtgt cacatgcctg tagtccctgc cacttgggag 51660 gctgaggtgg gaggttgaggctgcagtgag tcctgacccc accactgcac gccagcctgg 51720 atgacagagt gagacccaatctccaaagga agaaaaaaaa aaaaaaaaaa aaacattgaa 51780 caagaaagac atggttcttcaaagatttta gagtgtagtg gggagaaatg caggcacaga 51840 tttcaaaatg aagtggtaattactgggcat ttggagtacc tagagaagag ctgcgtagcc 51900 caagaaaacc atggcaagcccccacaatag gcagaggagc ccctaaagga gagactgaga 51960 tggaatggcc agaggaggagaaccaagaga ctgtagaaac atggagctca aaggaataga 52020 aacgaaattt caagaaagacttcagtggtc agatgaaaca ggaatgatga agggatacga 52080 gcccaggtgc ctagtggtgttcagcacaga gaagacaggc cgtgagtatg tgttgaccta 52140 cctggaagat catgtagccacattccagca tcttacaaag aagaaaactt cttgcgagaa 52200 agatgatttg ccctaacccacatagttctt agctacagtc ttagatttat tgatacctca 52260 cctaggacac taacaaagaaaaccaaagcc ataaaagtgt aaacaaatgg taggtagcac 52320 aacatttcat tatgtaaaaagttatgctga cattgacaac taggaaggga tattaacatg 52380 tatggcatac actttttgttccaaacatca tgctaagcac ttccacttat gttacctggt 52440 tctgcccctg aatgaaccctaagaaatagg tattctcctt tttatagaag agacacttgt 52500 gacactagcg gcctaatgtgacagagacac atgaggaatt cattgcaggg tgcccgggaa 52560 ataaatctta ctggggtttcaggagtcata ggttatcatt gctgctgtgt gatccttctc 52620 tggcaagcag agttactccattacaagaaa agactaaggg aacactaata acacatgtct 52680 ggagtataat tagacatagatccagaaagg aaagcgaatc atttcagaaa tgaatgtgtg 52740 cacttaagag tctgtttatagcatttagca gtttattaga ttttactggt tttgtcttgc 52800 aaattcagtt tgaggccgacagacgtgctt ttatcatcac tcttaactcc tttggcactg 52860 aattgagcaa agaagaccgagagaccctct atccaacctt cggcaaactc tatcctgagg 52920 ggttgcggct gttggctcaagcagaagact ttgaccagat gaagaacgta gcggatcatt 52980 acggagtatg tgatgacactggcttcccta agtcctttgt gttcattcat ttccatgtgc 53040 tttagaagct cacacatcaaatttccgctt atactcaaaa gaagtaataa tatttgtaag 53100 gtagcaatca gtgtttcttgctgaagcaat ctctccctaa gaaacacgtt aagtttctag 53160 cccaagctgc taatttttaaagttcctgcc aagcaagtac ttgtttaaat ctccaaggaa 53220 tggtcatctg agcactccttttgaagtgtt tgataaaaca agttccccca ggagactggt 53280 tcagtgttct ggaaatgtgagctcatatca acgttatgta aaatagtcta acccagcagc 53340 cctttcaggt acccactagggaaacaaggt tagccacaga acgtctaggt ggacggcaag 53400 gaacaatatg gatgtattagtttttccact caccttgctg agtggccaga aatcaggaga 53460 ggaaacatag ctgcttgtgtccttccccca ctccagcatc cagttgctcc ttggagcgcc 53520 tgaagctcct cagcttaaggttgtacactt tggctcagct tccattccct ctgacttgca 53580 cacatttaca taacctccaggtaacttttc cagggctaga atgacactca tcttttcctc 53640 cagatttctg aagcactccaggaaagatga aagtcagcaa atgcgtaacc cttgtgacat 53700 ctcatatgta attgcgtgagactctttagc gtaatattaa aatttgaatt tctcccaaat 53760 tccacccctt ccacactggaaactacatgt gaattttatc aattttcatc tcaaagaacc 53820 atgaaccctt actgaccaaatcatgaacat tcactgagct tctataatgt acctagctag 53880 ccctaagaac ctgagtgagaaaatgagctc aaacactgta tactgagagt tgaattgcct 53940 tgtagccctg aacaagccaataggctagaa tttcatccac actcttgtta tttggatttt 54000 tttattgttt cttcgggaggccaaaatcct ataaaaaaac attcagtcct cctggtattt 54060 ttttttaaat aggtacacaatttctataat gctaggaaaa atatatttat atacatttat 54120 aagaaatgaa aatgtctttaaaattaatgt ctttgttttt aaggtataca aacctttatt 54180 tgaagctgta ggtggcagtgggggaaagac attggaggac gtgttttacg agcgtgaggt 54240 atgatataag tggaaatactattagcttat ttttctctta ctgtggattt tatatatttt 54300 cttactttgg gttttctttttacagctgat catgccaatt gattaggcac attcatagct 54360 tgatagtcag tgatgtattattgctacaaa gttacacaca gacctaggaa tctttgtgct 54420 agcagtgctg ctaaagtttgtagcttttct ttcttttttt tttttttttt ataccctcct 54480 aaaagagttt acttaaaaactgcgagaagt aatggaagta cattcaattc ctcatggaat 54540 ttgctgaaga acattattacaaatgctaca aattttagtg taaaattttg gttaatcata 54600 tgagataatg tttcttttttaaaaaaaaaa ttgtgttata ctttaagttc tgagatacat 54660 gtgcagaatg tgcaggttttgttatgtagg tatacgtgtg ccatggtggg ttgctgcacc 54720 cgtcaaccca tcatctactttaggtatttc tcctaatgct atccctcccc tattcccccc 54780 accgacaggc cccagtgtgtgatgttcccc tccctgtgct catatgtttt cattgttcaa 54840 ctccccctta tgagtgaaaacatgtggtgt ttggtttttc tgttcctgtg ttagtttgct 54900 gagaatgatg gtctccagctttatccatgt ccctgcaaag gacatgaact catccttttt 54960 tatggctgca tagtattccatggtatatat gtgccacatt ttctttatcc aggctatcac 55020 tgatgggcat ttgggttggttccaagtctt tgctattggg aaaagtgctg caataaacat 55080 atgtgtgcag gtgtctttatagtaggatga tttataatct tttaggtata tacccagtaa 55140 tgggatcgct gggtcaaatgatatttctgg ttctagatcc ttgaagaatt gccacactat 55200 cttccacaat ggttgaactaatttacactc ccactaacag tgtgaaagcg ttcctatttc 55260 aatttggagc ttttcttgtgattctacaaa cttactttaa taacttccgt tgtctacatt 55320 tatctgcaga gtgacactctctctttagaa ctttctgagt tgtcaccata ctcctctatt 55380 tggcccaatt tagagattgcctgtttaaac acaaataagc actgcacaag ttcaagccag 55440 tcagtagctt aatattattccataatgttc caaataagtt gatgatctgt gttacttttt 55500 ttctttcctc aggtacaaatgaatgtgctg gcattcaaca gacagttcca ctacggtgtg 55560 ttttatgcat atgtaaagctgaaggaacag gaaattagaa atattgtgtg gatagcagaa 55620 tgtatttcac agaggcatcgaactaaaatc aacagttaca ttccaatttt ataacccaag 55680 taaggttctc aaatgtagaaaattataaat gttaaaagga agttattgaa gaaaataaaa 55740 gaaattatgt tatattatctagactacaca aaagtaagcc acactatatc ttcatgagtt 55800 gcaaatccat ggaaacacagtaaaccagcc ctgaaacaaa gcatttcctt gttttcagtg 55860 gtattagatc ttgtttccacatgtctgtct cattcttcac tgggccttac aggttagttt 55920 taattaactc tatggtatttttcttattct tgtttgatca tgttaaaaat tggacctaat 55980 aaaagtattt tattcttgcttttccatgct tctctacagg tccaaatact gaatgtctcc 56040 tttacttttt ctcttttaaatttttttcta gacagggtct cactctgtca cctaggctac 56100 agtgcagtgg tgtgatcacagctcactgca gcctcgactt cccaggctca agtgatcctc 56160 ccagctctca gcctccaaagtagctggcac tacaagtgta cacccccaca caaggctaag 56220 ttttgtattt tttgtagagacagggtttca acatattatc caggctggtg tcgaattcct 56280 gggctccagg gatccacagtcccccttggc ctcccaaagt gttgggatta catgcatgag 56340 ccactgtgct gggcttcatttacattttaa ctgtctgttc cttgcctaga ttcacagaaa 56400 tccaaagctg tatgtagtcaacatggttca caagtgttgg aaaatgtgtt ttttgttttg 56460 ttttgttttg ttttgtttcgttttgttttg agacagagtt tccctctgtc gcccaggcta 56520 gagtgcaatg gcgtgatctcggctcactgc aacctccacc tcccagattc aagcaactct 56580 ctgcctcagc ctcccgagtagctgggatta caagcaccca ccactacact cagctaattt 56640 tttgtatttt tagtagagccggggtttcac catcttggcc aggctgatct tgaactcctg 56700 agctcatgat ccacccgcctcagcctccca aagtgctggg attacaggcc ccttgttcag 56760 ccactgcacc tggccccttattttgttttt gttttctaat atactttgat gtaatcagct 56820 tgagaaagca acacaatttcaaatcctatc ttctagatgc aagcagtgct aaatttgtta 56880 ataaatttgc ttttcacacctttctttaaa taaaaggtat atctctcttt cttgtggttt 56940 cttccttctt tacatgagaagaaaatgacc tccttaattg tagtttactc attcaaaaat 57000 cgcaggttca gttttcagggtatgaattat ctattaaaat ctcagtagaa atggtgcata 57060 gagctaccag gaaaaaaaagacaacaaagc actgctcaag tccttccttt aaatataaaa 57120 catattaaca tattcctaaatgtaaaatct atatttgcca tgttttaaaa aataaaccca 57180 tatatatttg aaaattcttcaaatcagaat atgtacagag agtatacaaa tactgatatc 57240 tagaattaga tgcatagaccaaggagtaaa tcaaattttc ttcaaaaatc tagctttttt 57300 agttaaatat tttaataattttagtgatag gaactttttt gttgttgttg aaacagggtc 57360 ttgctccatc acccaggctggaatgcagtg gtgggatcac aggttactgg agcctccacc 57420 tcccaggctc aagtgatcctcctgcctctc agcctcccaa gtagctggaa ccagaggtgt 57480 gtgccaccac aactgcctaatttttttttt cttttttaag gtagagacag tgtctctaca 57540 tgttgcccag gctgatctcaaactcaggca atcctcccac ctcgacttcc ccaagtgcta 57600 ggattacagc cacaacgcccggccaaaagg aacttttaag catacagtaa aatagtggct 57660 catagaacca ggaagaaagccaagtgccta gcctcacat 57699 4 345 PRT Mus musculus 4 Glu Leu Tyr Phe AsnVal Asp Asn Gly Tyr Leu Glu Gly Leu Val Arg 1 5 10 15 Gly Met Lys AlaGly Val Leu Ser Gln Ala Asp Tyr Leu Asn Leu Val 20 25 30 Gln Cys Glu ThrLeu Glu Asp Leu Lys Leu His Leu Gln Ser Thr Asp 35 40 45 Tyr Gly Asn PheLeu Ala Asn Glu Ala Ser Pro Leu Thr Val Ser Val 50 55 60 Ile Asp Asp LysLeu Lys Glu Lys Met Val Val Glu Phe Arg His Met 65 70 75 80 Arg Asn HisAla Tyr Glu Pro Leu Ala Ser Phe Leu Asp Phe Ile Thr 85 90 95 Tyr Ser TyrMet Ile Asp Asn Val Ile Leu Leu Ile Thr Gly Thr Leu 100 105 110 His GlnArg Ser Ile Ala Glu Leu Val Pro Lys Cys His Pro Leu Gly 115 120 125 SerPhe Glu Gln Met Glu Ala Val Asn Ile Ala Gln Thr Pro Ala Glu 130 135 140Leu Tyr Asn Ala Ile Leu Val Asp Thr Pro Leu Ala Ala Phe Phe Gln 145 150155 160 Asp Cys Ile Ser Glu Gln Asp Leu Asp Glu Met Asn Ile Glu Ile Ile165 170 175 Arg Asn Thr Leu Tyr Lys Ala Tyr Leu Glu Ser Phe Tyr Lys PheCys 180 185 190 Thr Leu Leu Gly Gly Thr Thr Ala Asp Ala Met Cys Pro IleLeu Glu 195 200 205 Phe Glu Ala Asp Arg Arg Ala Phe Ile Ile Thr Ile AsnSer Phe Gly 210 215 220 Thr Glu Leu Ser Lys Glu Asp Arg Ala Lys Leu PhePro His Cys Gly 225 230 235 240 Arg Leu Tyr Pro Glu Gly Leu Ala Gln LeuAla Arg Ala Asp Asp Tyr 245 250 255 Glu Gln Val Lys Asn Val Ala Asp TyrTyr Pro Glu Tyr Lys Leu Leu 260 265 270 Phe Glu Gly Ala Gly Ser Asn ProGly Asp Lys Thr Leu Glu Asp Arg 275 280 285 Phe Phe Glu His Glu Val LysLeu Asn Lys Leu Ala Phe Leu Asn Gln 290 295 300 Phe His Phe Gly Val PheTyr Ala Phe Val Lys Leu Lys Glu Gln Glu 305 310 315 320 Cys Arg Asn IleVal Trp Ile Ala Glu Cys Ile Ala Gln Arg His Arg 325 330 335 Ala Lys IleAsp Asn Tyr Ile Pro Ile 340 345 5 345 PRT Bos taurus 5 Glu Leu Tyr PheAsn Val Asp Asn Gly Tyr Leu Glu Gly Leu Val Arg 1 5 10 15 Gly Leu LysAla Gly Val Leu Ser Gln Ala Asp Tyr Leu Asn Leu Val 20 25 30 Gln Cys GluThr Leu Glu Asp Leu Lys Leu His Leu Gln Ser Thr Asp 35 40 45 Tyr Gly AsnPhe Leu Ala Asn Glu Ala Ser Pro Leu Thr Val Ser Val 50 55 60 Ile Asp AspArg Leu Lys Glu Lys Met Val Val Glu Phe Arg His Met 65 70 75 80 Arg AsnHis Ala Tyr Glu Pro Leu Ala Ser Phe Leu Asp Phe Ile Thr 85 90 95 Tyr SerTyr Met Ile Asp Asn Val Ile Leu Leu Ile Thr Gly Thr Leu 100 105 110 HisGln Arg Ser Ile Ala Glu Leu Val Pro Lys Cys His Pro Leu Gly 115 120 125Ser Phe Glu Gln Met Glu Ala Val Asn Ile Ala Gln Thr Pro Ala Glu 130 135140 Leu Tyr Asn Ala Ile Leu Val Asp Thr Pro Leu Ala Ala Phe Phe Gln 145150 155 160 Asp Cys Ile Ser Glu Gln Asp Leu Asp Glu Met Asn Ile Glu IleIle 165 170 175 Arg Asn Thr Leu Tyr Lys Ala Tyr Leu Glu Ser Phe Tyr LysPhe Cys 180 185 190 Thr Leu Leu Gly Gly Thr Thr Ala Asp Ala Met Cys ProIle Leu Glu 195 200 205 Phe Glu Ala Asp Arg Arg Ala Phe Ile Ile Thr IleAsn Ser Phe Gly 210 215 220 Thr Glu Leu Ser Lys Glu Asp Arg Ala Lys LeuPhe Pro His Cys Gly 225 230 235 240 Arg Leu Tyr Pro Glu Gly Leu Ala GlnLeu Ala Arg Ala Asp Asp Tyr 245 250 255 Glu Gln Val Lys Asn Val Ala AspTyr Tyr Pro Glu Tyr Lys Leu Leu 260 265 270 Phe Glu Gly Ala Gly Ser AsnPro Gly Asp Lys Thr Leu Glu Asp Arg 275 280 285 Phe Phe Glu His Glu ValLys Leu Asn Lys Leu Ala Phe Leu Asn Gln 290 295 300 Phe His Phe Gly ValPhe Tyr Ala Phe Val Lys Leu Lys Glu Gln Glu 305 310 315 320 Cys Arg AsnIle Val Trp Ile Ala Glu Cys Ile Ala Gln Arg His Arg 325 330 335 Ala LysIle Asp Asn Tyr Ile Pro Ile 340 345

That which is claimed is:
 1. An isolated peptide consisting of an aminoacid sequence selected from the group consisting of: (a) an amino acidsequence shown in SEQ ID NO: 2; (b) an amino acid sequence of an allelicvariant of an amino acid sequence shown in SEQ ID NO: 2, wherein saidallelic variant is encoded by a nucleic acid molecule that hybridizesunder stringent conditions to the opposite strand of a nucleic acidmolecule shown in SEQ ID NOS: 1 or 3; (c) an amino acid sequence of anortholog of an amino acid sequence shown in SEQ ID NO: 2, wherein saidortholog is encoded by a nucleic acid molecule that hybridizes understringent conditions to the opposite strand of a nucleic acid moleculeshown in SEQ ID NOS: 1 or 3; and (d) a fragment of an amino acidsequence shown in SEQ ID NO: 2, wherein said fragment comprises at least10 contiguous amino acids.
 2. An isolated peptide comprising an aminoacid sequence selected from the group consisting of: (a) an amino acidsequence shown in SEQ ID NO: 2; (b) an amino acid sequence of an allelicvariant of an amino acid sequence shown in SEQ ID NO: 2, wherein saidallelic variant is encoded by a nucleic acid molecule that hybridizesunder stringent conditions to the opposite strand of a nucleic acidmolecule shown in SEQ ID NOS: 1 or 3; (c) an amino acid sequence of anortholog of an amino acid sequence shown in SEQ ID NO: 2, wherein saidortholog is encoded by a nucleic acid molecule that hybridizes understringent conditions to the opposite strand of a nucleic acid moleculeshown in SEQ ID NOS: 1 or 3; and (d) a fragment of an amino acidsequence shown in SEQ ID NO: 2, wherein said fragment comprises at least10 contiguous amino acids.
 3. An isolated antibody that selectivelybinds to a peptide of claim
 2. 4. An isolated nucleic acid moleculeconsisting of a nucleotide sequence selected from the group consistingof: (a) a nucleotide sequence that encodes an amino acid sequence shownin SEQ ID NO: 2; (b) a nucleotide sequence that encodes of an allelicvariant of an amino acid sequence shown in SEQ ID NO: 2, wherein saidnucleotide sequence hybridizes under stringent conditions to theopposite strand of a nucleic acid molecule shown in SEQ ID NOS: 1 or 3;(c) a nucleotide sequence that encodes an ortholog of an amino acidsequence shown in SEQ ID NO: 2, wherein said nucleotide sequencehybridizes under stringent conditions to the opposite strand of anucleic acid molecule shown in SEQ ID NOS: 1 or 3; (d) a nucleotidesequence that encodes a fragment of an amino acid sequence shown in SEQID NO: 2, wherein said fragment comprises at least 10 contiguous aminoacids; and (e) a nucleotide sequence that is the complement of anucleotide sequence of (a)-(d).
 5. An isolated nucleic acid moleculecomprising a nucleotide sequence selected from the group consisting of:(a) a nucleotide sequence that encodes an amino acid sequence shown inSEQ ID NO: 2; (b) a nucleotide sequence that encodes of an allelicvariant of an amino acid sequence shown in SEQ ID NO: 2, wherein saidnucleotide sequence hybridizes under stringent conditions to theopposite strand of a nucleic acid molecule shown in SEQ ID NOS: 1 or 3;(c) a nucleotide sequence that encodes an ortholog of an amino acidsequence shown in SEQ ID NO: 2, wherein said nucleotide sequencehybridizes under stringent conditions to the opposite strand of anucleic acid molecule shown in SEQ ID NOS: 1 or 3; (d) a nucleotidesequence that encodes a fragment of an amino acid sequence shown in SEQID NO: 2, wherein said fragment comprises at least 10 contiguous aminoacids; and (e) a nucleotide sequence that is the complement of anucleotide sequence of (a)-(d).
 6. A gene chip comprising a nucleic acidmolecule of claim
 5. 7. A transgenic non-human animal comprising anucleic acid molecule of claim
 5. 8. A nucleic acid vector comprising anucleic acid molecule of claim
 5. 9. A host cell containing the vectorof claim
 8. 10. A method for producing any of the peptides of claim 1comprising introducing a nucleotide sequence encoding any of the aminoacid sequences in (a)-(d) into a host cell, and culturing the host cellunder conditions in which the peptides are expressed from the nucleotidesequence.
 11. A method for producing any of the peptides of claim 2comprising introducing a nucleotide sequence encoding any of the aminoacid sequences in (a)-(d) into a host cell, and culturing the host cellunder conditions in which the peptides are expressed from the nucleotidesequence.
 12. A method for detecting the presence of any of the peptidesof claim 2 in a sample, said method comprising contacting said samplewith a detection agent that specifically allows detection of thepresence of the peptide in the sample and then detecting the presence ofthe peptide.
 13. A method for detecting the presence of a nucleic acidmolecule of claim 5 in a sample, said method comprising contacting thesample with an oligonucleotide that hybridizes to said nucleic acidmolecule under stringent conditions and determining whether theoligonucleotide binds to said nucleic acid molecule in the sample.
 14. Amethod for identifying a modulator of a peptide of claim 2, said methodcomprising contacting said peptide with an agent and determining if saidagent has modulated the function or activity of said peptide.
 15. Themethod of claim 14, wherein said agent is administered to a host cellcomprising an expression vector that expresses said peptide.
 16. Amethod for identifying an agent that binds to any of the peptides ofclaim 2, said method comprising contacting the peptide with an agent andassaying the contacted mixture to determine whether a complex is formedwith the agent bound to the peptide.
 17. A pharmaceutical compositioncomprising an agent identified by the method of claim 16 and apharmaceutically acceptable carrier therefor.
 18. A method for treatinga disease or condition mediated by a human transporter protein, saidmethod comprising administering to a patient a pharmaceuticallyeffective amount of an agent identified by the method of claim
 16. 19. Amethod for identifying a modulator of the expression of a peptide ofclaim 2, said method comprising contacting a cell expressing saidpeptide with an agent, and determining if said agent has modulated theexpression of said peptide.
 20. An isolated human transporter peptidehaving an amino acid sequence that shares at least 70% homology with anamino acid sequence shown in SEQ ID NO:
 2. 21. A peptide according toclaim 20 that shares at least 90 percent homology with an amino acidsequence shown in SEQ ID NO:
 2. 22. An isolated nucleic acid moleculeencoding a human transporter peptide, said nucleic acid molecule sharingat least 80 percent homology with a nucleic acid molecule shown in SEQID NOS: 1 or
 3. 23. A nucleic acid molecule according to claim 22 thatshares at least 90 percent homology with a nucleic acid molecule shownin SEQ ID NOS: 1 or 3.